@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix dc: . @prefix skos: . vivo:departmentOrSchool "Medicine, Faculty of"@en, "Pathology and Laboratory Medicine, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Melo, Maico DaPonte"@en ; dcterms:issued "2009-06-26T22:30:47Z"@en, "1999"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description """Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, is a facultative intracellular pathogen capable of survival in macrophages. Several macrophage receptors including complement receptor type 3 (CR3, CDI lb/CD 18) are reportedly capable of binding MTB. It has been suggested that MTB survival in macrophages is determined by the receptor to which MTB binds before being phagocytosed. A CDI lb knockout mouse model was used as a source of macrophages to test whether CR3 is essential for the association of MTB with macrophages and to examine whether the absence of CR3 on macrophages alters the intracellular fate of MTB. Together, these research objectives were aimed at improving our understanding of the role CR3 plays in the pathogenesis of tuberculosis. Studies using both alveolar macrophages (AM) and peritoneal macrophages (PM) showed CR3 was not essential for MTB binding by macrophages, either in the presence or absence of serum. However, CR3 was involved in the association of MTB with macrophages in the absence of serum and was important for the efficient binding of MTB under conditions where serum was present. Compared to macrophages expressing CDI lb (CDI lb+/+), CDI lb-knockout (CDI lb-/-) macrophages were less able to associate with MTB, either opsonically or non-opsonically. Under opsonic conditions, the enhanced binding of MTB to macrophages was mediated by a heat-labile serum component, as heat-inactivation of serum abrogated the increased binding. Using immunoglobulin-deficient serum to opsonize MTB demonstrated that the increased binding of MTB was not mediated by immunoglobulins. The role of either the classical or alternative complement pathways in mediating the increased binding of MTB by PM was also investigated. The observations made here indicate that, in the presence of low serum concentrations, increased binding of MTB by CDI lb+/+ PM is mediated predominantly via activation of the classical pathway but is independent of immunoglobulins. The intracellular survival and replication of MTB following phagocytosis by either CDI lb-/- PM or CDI lb+/+ PM was investigated and no significant difference in the replication of MTB was detected where similar numbers of MTB were ingested. Therefore, the observations made here do not support the hypothesis that MTB exploits CR3 as a means to evade being killed by macrophages. As an alternative approach to the CDI lb knockout mouse model, an alveolar macrophage cell line, MH-S, was tested for its suitability as a model to investigate the role of CR3 in mediating the interaction of MTB with macrophages. Initially, the characterization of MH-S binding properties was performed. However, the principle objective with the MH-S cell line was to isolate CR3-negative populations of MH-S as a means to investigate the role of CR3 in the association of MTB. MTB association assays showed that the association of MTB with MH-S cells was comparable with mouse AM. In the presence of serum, increased MTB binding by MH-S cells was also mediated by a heatlabile serum component and competitive inhibition assays using anti-CR3 monoclonal antibodies showed that CR3 on MH-S cells is a quantitatively important receptor for MTB. However, attempts to isolate stable CR3-negative populations of MH-S cells using immunoselection were not successful and therefore, I was unable to use the MH-S cell line as a model to further characterize the role of CR3 in MTB pathogenesis."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/9724?expand=metadata"@en ; dcterms:extent "6199735 bytes"@en ; dc:format "application/pdf"@en ; skos:note "Role of Complement Receptor Type 3 in M y c o b a c t e r i u m tuberculosis Pathogenesis by Maico DaPonte Melo B.Sc , The University of Victoria, 1996 A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in The Faculty of Graduate Studies Department of Pathology and Laboratory Medicine We accept this thesis as conforming to the required standard / The University of British Columbia June 1999 © Maico DaPonte Melo, 1999 in presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, 1 agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Tofko /o 0.05). 31 1600 Zymosan EIgG ElgMC Figure 3. Binding of Zymosan, EIgG, and E l g M C ' to P M . P M obtained from either C D I lb+/+ or C D I lb-/- mice were adhered to coverslips in serum-supplemented medium for 18 hours. The ability of these macrophages to bind Zymosan, EIgG, and E l g M C in a serum-free environment for 3 hours was then tested. Binding was assessed as the number 6f particles associated with 100 macrophages (Association Index). The mean + S E M is shown for n = 6 (3 separate experiments with duplicate coverslips in each experiment). 32 3.4 Association of MTB with PM from CDllb-/- and CDllb+/+ mice. 3.4.1 Non-opsonic Association of MTB with PM Under non-opsonic conditions, the association of M T B with C D l l b - / - P M was significantly less than that observed for CDI lb+/+ P M (P < 0.008 for all comparisons). The percentage of CDI lb-/- P M associated with 1 or more, greater than 10, or greater than 30 bacteria per macrophage was 60 + 4.4%, 13.7 + 2.5%, 0.3 + 0.2%, respectively (Figure 4). Whereas, the percentage of CDI lb+/+ P M associated with 1 or more, greater than 10, or greater than 30 bacteria per macrophage was 73 + 2.5%, 28 + 2.7%, and 2+1%, respectively (Figure 4). 1 0 0 1 or more > 10 bacteria > 30 bacteria Figure 4. Non-opsonic association of M T B with CDI lb+/+ P M and CDI lb-/- P M . P M obtained from either CDI lb+/+ or CDI lb-/- mice were adhered to coverslips in serum-supplemented medium for 18 hours. The ability of these macrophages to bind M T B in a serum-free environment was tested. The mean percentage + S E M of the macrophages binding 1 or more, greater than 10, and greater than 30 bacteria is reported for n = 12 (6 separate experiments with duplicate coverslips from each experiment). 33 3.4.2 Opsonic Association of MTB with PM Similar to previously reported observations, the binding of M T B by P M was enhanced by the addition of serum [18]. In the presence of serum, there was a more dramatic difference between the association of M T B with CDI lb-/- P M and with CDI lb+/+ P M than that seen under non-opsonic conditions (compare Figures 4 and 5). In the presence of 1% NMS, 88 + 2.3% of CDI lb-/- P M associated with one or more bacterium whereas 100 + 0.2% of CDI lb+/+ P M were observed to associate with one or more bacterium (Figure 5; P < 0.001 at the level of binding at least one bacterium). The percentage of P M associated with greater than 10 bacteria were 51 + 4.6% and 98 ± 3.3% for CDI lb-/- P M and CDI lb+/+ P M , respectively (Figure 5; P < 0.001 at the greater than 10 bacteria level). CDI lb-/- P M were approximately 9x less efficient at binding M T B than CDI lb+/+ P M with 6.2 + 2.3% and 45 + 3.3% of macrophages binding greater than 30 bacteria, respectively (Figure 5; P < 0.001 at the greater than 30 bacteria per macrophage level). 34 1 or more >10 bacteria >30 bacteria Figure 5 . Opsonic association of M T B with CDI lb+/+ P M and CDI lb-/- P M . P M obtained from either C D I lb+/+ or C D I lb-/- mice were adhered to coverslips in serum-supplemented medium for 18 h. The ability of these macrophages to bind M T B in the presence of 1% normal mouse serum (NMS) was tested. The mean percentage + S E M of the macrophages binding 1 or more, greater than 10, or greater than 30 bacteria is reported for n = 12 (6 separate experiments with duplicate coverslips from each experiment). Note: the S E M for CDI lb+/+ P M binding M T B at the level of 1 or more M T B is 0.2%. 35 3.4.3 Effects of heat-inactivated and immunoglobulin-negative serum on association of MTB with PM. Heat-inactivated normal mouse serum and RAG-1 (-/-) mouse serum were used to investigate further the enhanced binding of M T B by P M in the presence of serum opsonins. Heat-inactivation (56°C for 30 minutes) of serum abrogates activation of complement and other serum components which are sensitive to the heat treatment [53]. R A G serum, obtained from mice which do not possess differentiated T and B cells, is immunoglobulin-deficient (IgG-negative status confirmed by ELISA, Appendix I) and therefore provides a means to investigate the contribution of natural antibody in mediating the association of M T B with macrophages. R A G serum also provides a means to evaluate the role antibody-mediated complement pathway activation plays in enhancing the association of M T B with macrophages. In the presence of heat-inactivated serum, the binding of M T B by P M of either phenotype was significantly less than that seen when normal mouse serum was used (P < 0.00005) and was similar to levels of M T B binding seen under non-opsonic conditions (Figure 6). When NMS was used to opsonize M T B , approximately half of the CDI lb-/- P M and nearly all CDI lb+/+ P M were associated with greater than 10 bacteria (Figure 6). However, when heat-inactivated serum is used, less than 20% of CDI lb-/- P M and only 33 + 3.4 % of CDI lb+/+ P M associate with greater than 10 bacteria (Figure 6). The observed abrogation of enhanced binding following heat treatment of serum is in agreement with previous reports [6,18] and is additional evidence supporting the role of complement proteins in mediating M T B binding under conditions where serum is present. 36 For P M of either phenotype, no appreciable difference in the binding of M T B was observed when R A G serum was compared to NMS (Figure 6; P > 0.05 for M T B binding at all levels). In the presence of 1% R A G serum, the percentage of CDllb+/+ P M associated with 1 or more, greater than 10, or greater than 30 bacteria per macrophage was 98 + 5.1, 92 + 5.1, and 35.3 ± 3.1, respectively (Figure 6). For M T B binding to CDI lb-/- P M in the presence of 1% R A G serum, the percentage of cells with 1 or more, greater than 10, or greater than 30 bacteria per macrophage was 88 + 3.2, 58 + 4.0, and 3.2 + 1.0, respectively (Figure 6). Under the conditions used here, the observations indicate that enhanced binding of M T B seen in the presence of serum is independent of immunoglobulins. 37 1 or more >10 bacteria >30 bacteria 1 or more >10 bacteria >30 bacteria Figure 6. Effects of heat-inactivated and immunoglobulin-negative serum on binding of M T B to CDI lb+/+ and CDI lb-/- P M . P M obtained from either CDI lb+/+ or CDI lb-A-mice were adhered to coverslips in serum-supplemented medium for 18 hours. The ability of these macrophages to bind M T B either in the absence (-) or presence of mouse sera was tested. The sera used in these binding assays were normal mouse serum (NMS), heat-inactivated (HI) normal mouse serum, and serum deficient for immunoglobulins (RAG), all at a concentration of 1 % in binding medium. The mean percentage + S E M of the macrophages binding 1 or more, greater than 10, and greater than 30 bacteria is reported for n = 6 (3 separate experiments with duplicate coverslips from each experiment). 38 3.4.4 Association of pre-opsonized MTB versus association of MTB in the presence of serum with PM. It has been reported previously that, compared to serum-free binding, the binding of M T B by human monocytes is enhanced when M T B is either pre-opsonized with serum or when serum is added to the binding medium [6]. However, to determine whether the presence of serum in the binding medium had an effect on the enhanced binding of M T B that could not be explained solely by M T B opsonization, a comparison of M T B binding between pre-opsonized M T B and M T B in the presence of serum was performed. In this series of experiments, M T B binding to either CDI lb+/+ P M or CDI lb-/- P M was assessed in assays testing either NMS or R A G serum (Table 1). The observations indicate that serum mediated binding of M T B by P M was comparable whether M T B was pre-opsonized with serum or binding occurred in the presence of serum. No significant difference in the percentage of CDI lb+/+ P M binding M T B was observed between assays in which M T B was either pre-opsonized with serum or incubated with serum (P > 0.05 for all conditions and at all levels of M T B binding). In the presence of NMS or R A G serum, approximately half of the CDI lb+/+ P M in the monolayer were associated with greater than 30 M T B (Table 1). Similarly, when M T B was pre-opsonized with either NMS or R A G seriim, approximately half of the CDI lb+/+ P M were associated with greater than 30 M T B (Table 1). Consistent with observations reported in previous sections, the binding of M T B by CDI lb-/- P M was significantly less than that observed for CDI lb+/+ P M (P < 0.0013 for all conditions tested). Similar to CDI lb+/+ P M , there was also no significant difference in M T B binding to CDI lb-/- P M as a result of pre-opsonization or incubation of M T B with either NMS or R A G serum (P > 0.05 at all levels 39 of M T B binding). In the presence of serum or following pre-opsonization with serum approximately 40% of CDI lb-/- P M were observed to associate with greater than 10 M T B whereas, less than 5% of CDI lb-/- P M were observed to associate with M T B at the greater than 30 level (Table 1). Table 1. Association of MTB with PM: A comparison between pre-opsonized MTB and MTB in the presence of serum . Percent Macrophages Binding MTB CDllb+/+ CDllb-/-Bacteria/ macrophage 0 1- 10 11 -30 >30 0 1-10 11-30 >30 NMS 1.8 ± 0.8 1.6 ± 1.2 35 ± 4.3 62 ± 3.6 14 ± 1 . 9 46 ± 5 . 1 37 ± 4 . 5 3.8 ± 2 . 8 Pre-NMS 1.0 ± 0.7 2.1 ± 1.0 38 ± 2.6 60 ± 3 . 2 19 ± 2 . 6 40 ± 3 . 4 39 ± 3 . 2 3.3 ± 1.4 R A G 1.5 ± 0.5 1.6 ± 0 . 8 37 ± 2.8 60 ± 3 . 2 15 ± 1.7 46 ± 4 . 1 39 ± 4 . 0 1.0 ± 0 . 6 Pre-RAG 2.3 ± 1.1 4.3 ± 2 . 2 44 ± 2.5 50 ± 3 . 4 16 ± 1.8 46 ± 4 . 6 37 ± 3 . 1 l.i 3 ± 0 . 6 Association of MTB in the presence of 1% normal mouse serum (NMS) or 1% RAG mouse serum (RAG) and association of MTB pre-opsonized with 10% normal mouse serum (Pre-NMS) or pre-opsonized with 10% RAG mouse serum (Pre-RAG) was assessed. The mean + SEM for n = 4 (2 separate experiments with duplicate coverslips in each experiment is reported). Binding of MTB was measured as the percentage of PM in the monolayer that bound 0, 1-10, 11-30, or greater than 30 bacteria. 40 3.4.5 Role of Complement in the Association of M T B with P M . Thus far, the results obtained indicated that the enhanced binding of M T B to P M in the presence of serum was due to a heat-labile serum component present in immunoglobulin-negative and immunoglobulin-positive serum, suggesting complement activation and fixation that was independent of serum immunoglobulins. The role of complement in mediating enhanced M T B binding by macrophages has been studied extensively [6,16,54]; however, the precise mechanism by which M T B activates complement is still not fully understood. Hetland et al. reported that anti-LAM immunoglobulins play a significant role in mediating the activation of the classical complement pathway and suggested that this was a means for M T B to increase its uptake by macrophages [54]. On the other hand, Schlesinger et al. reported that M T B activates the alternative complement pathway through interactions of M T B surface molecules with complement proteins [6]. Recently, using human monocytes and equine serum, Schorey et al. reported that M T B is capable of scavenging C2a to create a novel C3 convertase that leads to deposition of C3b on the M T B surface and enhanced uptake of M T B by human monocytes [16]. Due to the recent developments regarding the role of complement in mediating enhanced M T B binding by macrophages and because the role of complement in these interactions is not fully understood, I was interested in investigating whether a specific activation pathway of complement, either the classical or alternative pathway, was responsible for the serum-mediated enhanced binding of M T B that I had observed in my studies. Enhanced M T B binding resulting from the activation of either complement pathway is believed to be due to the deposition of opsonic C3 proteins on M T B . Both the classical and alternative 41 pathways are dependent upon M g 2 + for the formation of their respective C3 convertases, C4b2a and C3bBb [55-57]. However, the classical pathway differs from the alternative pathway in that it is dependent upon C a 2 + for the formation of the Clqrs complex [57,59]. E D T A which chelates both C a 2 + and M g 2 + is therefore reported to be a means to inactivate both the classical and alternative pathways of complement whereas E G T A which chelates C a 2 + but not M g 2 + is a means to inactivate the classical pathway while leaving the alternative pathway intact [6,55]. In addition to using chelating agents to investigate the role of complement in mediating M T B binding in the presence of serum, I used factor B-depleted and C2-depleted human serum to assess the roles of either the alternative or classical pathways, respectively. For these experiments, pooled human serum (PHS) from healthy PPD-negative volunteers was used as the control serum because neither factor B-depleted nor C2-depleted mouse sera were commercially available at the time of these studies. It has been shown previously that for mouse P M , the serum-mediated enhanced binding of M T B occurs when either normal human serum or normal mouse serum are employed (unpublished observations Stokes laboratory). In the current series of experiments, the enhanced binding of M T B by P M seen with NMS was also observed with PHS, confirming that opsonins derived from human serum are compatible with the mouse model employed in these experiments. In the presence of either E D T A or EGTA, the association of M T B with P M dropped to levels below those seen with PHS and were also considerably lower than levels of M T B binding seen under serum-free conditions (Table 2). Less than 6% of CDI lb+/+ P M were observed to associate with M T B at the greater than 10 bacteria per 42 macrophage level when either chelating agent was present in the binding medium (Table 2). In contrast, nearly all CDI lb+/+ P M associated with greater than 10 bacteria in the presence of 1% PHS and under serum-free conditions 61 + 5.4 % of the CDI lb+/+ P M monolayer associated with greater than 10 bacteria (Table 2). The effects of E D T A and E G T A were similar for CDI lb-/- P M (Table 2). Based on these observations, it appears that EDTA and E G T A have much broader effects on M T B binding than complement pathway inactivation. To further assess the contribution of either the alternative or classical pathways in mediating the enhanced binding of M T B to macrophages, factor B-depleted and C2-depleted serum were used as a source of opsonins. Compared to PHS, the association of M T B with CDI lb+/+ P M was less when either factor B-depleted or C2-depleted serum were used as a source of opsonins (Table 2). However, the difference seen with C2-depleted serum was more dramatic. In the presence of 1% C2-depleted serum, the percentage of CDI lb+/+ P M associated with 1 or more, greater than 10, or greater than 30 bacteria was 90 + 8.6%, 63 ± 4.1%, and 6.0 ± 1.7%, respectively (Table 2). There was approximately 8x less macrophages associated with M T B at the greater than 30 level when C2-depleted serum was used instead of PHS as a source of opsonins. On the other hand, substituting factor B-depleted serum for PHS lead to a moderate decrease in M T B binding with approximately a two-fold reduction in the percentage of macrophages associated with greater than 30 bacteria (Table 2). Under the conditions tested, it appears that the enhanced binding of M T B to CDI lb+/+ P M is mediated predominantly through activation of the classical complement pathway. 43 Unlike the CDI lb+/+ P M , the role of either the alternative or classical pathways in mediating M T B binding to CDI lb-/- P M appears equivalent. In the presence of 1% C2-depleted serum, the percentage of CDI lb-/- P M associated with 1 or more, greater than 10, or greater than 30 bacteria was 65 ± 7.0%, 24 + 4.3%, and 2.0 + 0.9%, respectively (Table 2). When factor B-depleted serum was substituted for PHS, the percentage of cells binding 1 or more, greater than 10, or greater than 30 bacteria was 71 + 9.0%, 28 + 5.7%, and 1.5 + 0.6%, respectively (Table 2). Compared to PHS, there was approximately a two-fold reduction in the percentage of macrophages binding M T B at the greater than 10 bacteria per macrophage level when either factor B-depleted or C2-depleted serum was used as a source of opsonins. However, based on the observation that CDI lb is required for efficient serum mediated binding of M T B by macrophages, the use of CDI lb-/- P M to investigate the contribution of either the classical or alternative pathways introduces a degree of complexity that makes the data somewhat equivocal. 44 Table 2. Role of Complement in the Association of M T B with P M Percent Macrophages Binding M T B CDllb+/+ C D l l b - / -Bacteria /macrophage 0 1-10 11-30 >30 0 1 -10 11-30 >30 No serum 12 + 2.2 27 ± 2.2 49 ± 4.2 1 2 ± 1.2 41 ± 5.8 38 ± 4 . 9 19 ± 2 . 0 1.7 ± 1.0 PHS 2.0 ± 0.4 2.8 ± 1.2 48 ± 3.7 47 ± 3.5 17 ± 5.2 22 ± 2 . 7 51 ± 3 . 9 10 ± 3 . 4 PHS+EDTA 58 + 4.1 36 + 4.0 5.0 ± 3 . 2 0 ± 0 59 ± 5.5 38 ± 4 . 7 2.8 ± 1.4 0 ± 0 PHS+EGTA 58 + 3.5 40 ± 4.1 2.5 ± 1 0 ± 0 53 ± 6.9 41 ± 5 . 2 6.2 ± 2 . 6 0 ± 0 Factor B (-) 3.3 + 1.2 8.0 ± 2.8 63 ± 1.7 26 ± 3 . 2 29 ± 3.9 43 ± 3 . 3 27 ± 5 . 1 1.5 ± 1 C2 (-) 9.7 + 1.0 28 ± 4.5 57 ± 2.4 6.0 ± 1.7 36 ± 4.7 40 ± 2 . 7 22 ± 3.4 2.0 ± 0 . 9 Association of M T B in the presence of binding medium alone [No serum], 1% pooled human serum [PHS], 1% PHS with lOmM E D T A [PHS+EDTA] or 1% PHS with lOmM E G T A [PHS+EGTA] and in the presence of either 1% Factor B depleted human serum [Factor B(-)] or C2 depleted human serum [C2(-)] was assessed. The mean + S E M for n = 6 (3 separate experiments each with duplicate coverslips is reported). Binding of M T B was measured as the percentage of P M in the monolayer that bound 0, 1-10, 11-30, or greater than 30 bacteria. 3.5 Association of Zymosan, EIgG, and E l g M C with A M from C D l l b - / - and CDllb+/+ mice. Control particles were also used to probe for functional receptors on A M . Under the conditions used here, no significant difference in the association of zymosan, EIgG, or E l g M C was observed between CDI lb-/- A M and CDI lb+/+ A M (Figure 7; P = 0.25, 0.18, and 0.95 for zymosan, EIgG, and E l g M C , respectively). Both CDI lb-/- A M and CDI lb+/+ A M associated with zymosan very efficiently, with association indices of 2062 45 + 76 and 2203 + 82, respectively (Figure 7). The levels of zymosan binding are similar to those reported previously for Day 4 mouse A M [18]. As reported previously, A M did not bind EIgG as avidly as zymosan [18]. Here the association indices of EIgG for CDI lb-/-A M and CDI lb+/+ A M were 1702 ± 154 and 1322 ± 210, respectively (Figure 7). Similar to the observations with P M , no significant difference in E l g M C binding was observed for CDI lb-/- A M and CDI lb+/+ A M (P > 0.05), with association indices of 425 ± 77 and 433 + 102, respectively (Figure 7). 2500 Zymosan EIgG E l g M C Figure 7. Binding of Zymosan, EIgG, and E l g M C to A M . A M obtained from either CDI lb+/+ or CDI lb-/- mice were adhered to coverslips and cultured for 4 days. The ability of these macrophages to bind Zymosan, EIgG, and E l g M C in a serum-free environment for 3 hours was tested. Binding was assessed as the number of particles associated with 100 macrophages (Association Index). The mean + S E M is shown for n 6 (3 separate experiments with duplicate coverslips in each experiment). 46 3.6.1 Non-opsonic Association of M T B with A M Under non-opsonic conditions, the binding of M T B by CDI lb-/- A M was significantly less than that observed for CDI lb+/+ A M (P = 0.007 at the level of binding at least one bacterium). For CDI lb+/+ A M , 46 + 3.8% bound at least one bacterium whereas only 33.2 + 1.9% of CDI lb-/- A M were observed to bind at least one bacterium (Figure 8). At the level of 10 or more bacteria per macrophage, approximately 5% of CDI lb+/+ A M bound greater than 10 bacteria whereas only 1% of CDI lb-/- A M monolayers were observed to bind greater than 10 bacteria (Figure 8). 100 CQ CT C CQ CD11b+/+ AM CD11b- / - AM 1 or more > 10 bacteria> 30 bacteria Figure 8. Non-opsonic association of M T B with CDI lb+/+ A M and CDI lb-/- A M . A M obtained from either CDI lb+/+ or CDI lb-/- mice were adhered to coverslips and cultured for 4 days. The ability of these macrophages to bind M T B in a serum-free environment was tested. The mean percentage + S E M of the macrophages binding 1 or more, greater than 10, or greater than 30 bacteria is reported for n = 10 (5 separate experiments with duplicate coverslips from each experiment). 47 3.6.2 Opsonic Association of M T B with A M In the presence of normal mouse serum (NMS), M T B binding for either CDI lb-/- A M or CDI lb+/+ A M increased compared to binding under non-opsonic conditions (P < 0.008 for all comparisons between non-opsonic and opsonic binding for A M of either phenotype). Similar to non-opsonic conditions, M T B binding by CDI lb+/+ A M was significantly greater than that seen with CDI lb-/- A M (P = 0.003 and 0.004 at the level of binding at least 1 bacterium and greater than 10 bacteria, respectively). With 1% NMS present in the binding medium, 51.1 + 5.9% of CDI lb-/- A M bound at least 1 bacterium and 13 + 2.7% bound greater than 10 bacteria (Figure 9). The increased association of M T B with A M was more dramatic for CDI lb+/+ A M . In the presence of 1% NMS, 71 + 2.3% of CDI lb+/+ A M bound at least 1 bacterium and 25.2 ± 2.5% bound greater than 10 bacteria (Figure 9). 48 Figure 9. Opsonic association of M T B with CDI lb+/+ A M and CDI lb-/- A M . A M obtained from either C D I lb+/+ or CDI lb-/- mice were adhered to coverslips and cultured for 4 days. The ability of these macrophages to bind M T B in the presence of 1% normal mouse serum (NMS) was tested. The mean percentage + S E M of the macrophages binding 1 or more, greater than 10, or greater than 30 bacteria is reported for n = 10 (5 separate experiments with duplicate experiments from each experiment). 49 3.6.3 Effects of heat-inactivated and immunoglobulin-negative serum on the association of M T B with A M before and after in vitro differentiation. Heat-inactivated serum and R A G serum were again used to test the effects of serum on the enhanced binding of M T B by macrophages. Compared to NMS, the binding of M T B by Day 4 CDI lb+/+ A M was significantly less when HI was used a source of opsonins (P < 0.005 at all levels of M T B binding). For Day 4 CDI lb+/+ A M , the percentage of macrophages binding 1 or more, greater than 10, or greater than 30 bacteria per macrophage in the presence of 1% HI was 42 + 1.7%, 2.5 + 1.0%, and 0%, respectively (Figure 10). Likewise, M T B binding by Day 4 CDI lb-/- A M was significantly less when HI was used as a source of opsonins instead of NMS (P < 0.007 at all levels of M T B binding). The percentage of Day 4 CDI lb-/- A M binding 1 or more, greater than 10, or greater than 30 bacteria in the presence of 1% HI was 35 ± 5.9%, 0.3 + 0.3%, and 0%, respectively (Figure 10). For A M of either phenotype the binding of M T B in the presence of HI was comparable to levels seen under non-opsonic conditions (Figure 10). The observations made here are in agreement with a previous report [18] and provide additional evidence supporting the role of complement proteins in mediating the enhanced binding of M T B under conditions where serum is present. Similar to the results obtained with P M , no significant difference in the binding of M T B was observed for A M of either phenotype when R A G serum was used instead of NMS as a source of opsonins in binding assays (P > 0.05 at all levels of M T B binding). In the presence of 1% R A G serum, the percentage of Day 4 CDI lb+/+ A M associated with 1 or more, greater than 10, or greater than 30 bacteria per macrophage was 85 + 1.7%, 48 + 50 5.4%, and 12 ± 3.9%, respectively (Figure 10). For M T B binding to Day 4 CDI lb-/- A M in the presence of 1% R A G serum, the percentage of macrophages associated with 1 or more, greater than 10, or greater than 30 bacteria was 75 + 1.9%, 31 + 6.1%, and 3.8 + 1.8%, respectively (Figure 10). Therefore, similar to the observations with P M , the enhanced binding of M T B by A M of either phenotype appears to be mediated by a heat-labile serum component but is independent of immunoglobulins. In addition to testing the effects of serum on M T B binding, I also examined the effects of in vitro differentiation of A M on M T B binding. Previous studies have shown that CR3 expression, along with the expression of several other receptors is low for Day 0 A M but culturing of A M in vitro leads to an increase in CR3, and other receptor expression, by Day 4 A M [18]. Based on this observation, I was interested in testing the hypothesis that increased M T B binding by Day 4 A M was due to an increase in CR3 expression. The association of M T B with Day 0 and Day 4 A M from either CDI lb-/- or CDI lb+/+ mice was assessed. Presumably, if increased expression of CR3 was exclusively responsible for the enhanced binding of M T B by Day 4 A M , an increase in M T B binding would be observed with CDI lb+/+ A M differentiation but not with CDI lb-/- A M . However, in this series of experiments the association of M T B with A M of either phenotype increased with in vitro differentiation. Both CDI lb+/+ and CDI lb-/- Day 4 A M were capable of binding M T B more efficiently than their Day 0 counterparts (Figure 10). In the presence of NMS, the binding of M T B by Day 0 CDI lb+/+ A M was significantly less that that observed for Day 4 CDI lb+/+ A M (P = 0.001 at the level of binding at least one bacterium). The percentage of Day 0 CDI lb+/+ A M binding 1 or more, greater than 10, or greater than 30 51 bacteria in the presence of NMS was 58 + 4.0%, 7.8 ± 3.5%, and 0.3 ± 0.3%, respectively (Figure 10). Likewise, M T B binding in the presence of NMS by Day 0 CDI lb-/- A M was significantly less than that observed with Day 4 CDI lb-/- A M (P < 0.001). The percentage of Day 0 CDI lb-/- A M binding 1 or more, greater than 10, or greater than 30 M T B was 41 + 1.6%, 1.8 ± 0.7%, and 0%, respectively (Figure 10). The increased binding of M T B by Day 4 A M was also observed under non-opsonic conditions (Figure 10). Therefore, based on these observations the serum-mediated increased binding of M T B that accompanies differentiation cannot be attributed exclusively to CR3. In fact, the data suggest that the majority of M T B binding by A M , either in the presence or absence of serum, is mediated by receptors other than CR3. 52 1 or more > 10 > 30 1 or more > 10 Bacteria Per Macrophage > 30 Figure 10. Effects of heat-inactivated and immunoglobulin-negative serum on the association of M T B with A M before and after in vitro differentiation. A M obtained from either CDI lb+/+ or CDI lb-/- mice were adhered and cultured for 2 hours (Day O) or 4 days (Day 4). The ability of these macrophages to bind M T B either in the absence (-) or presence of mouse sera was assessed. The sera used in these binding assays were normal mouse serum (NMS), heat-inactivated normal mouse serum (HI), or serum deficient in immuno-globulins (RAG). A l l sera used at 1% concentrations in binding medium. The mean percentage + S E M of the macrophages binding 1 or more, greater than 10, or greater than 30 bacteria is reported for n = 4 (2 separate experiments with duplicate coverslips from each experiment). 53 3.6.4 Comparison of M T B binding between C 5 7 B L / 6 and B A L B / c Day 0 A M In addition to demonstrating that receptors other than CR3 play a quantitatively important role in the binding of M T B by A M , the in vitro differentiation experiments also identified that Day 0 A M from either CDI lb+/+ or CDI lb-/- mice respond to the addition of serum with increased M T B binding (Figure 10). The latter observation is in contrast to previous reports which found that Day 0 A M from B A L B / c do not respond to the addition of serum with enhanced binding of M T B [18]. In the present studies, the binding of M T B in the presence of 1% NMS by either CDI lb+/+ or CDI lb-/- Day 0 A M was significantly greater than M T B binding under non-opsonic conditions (P = 0.007 and 0.046 for Day 0 CDI lb+/+ A M and Day 0 CDI lb-/- A M , respectively). Because Day 0 A M from either CDI lb+/+ or CDI lb-/- mice appeared to behave differently with respect to M T B binding in the presence of serum than Day 0 A M from B A L B / c mice, I was interested in investigating this difference. The association of M T B with Day 0 A M obtained from CDI lb+/+ mice of C57BL/6 background was compared with Day 0 A M from B A L B / c mice. In the absence of serum, Day 0 A M from either C57BL/6 mice or B A L B / c mice behaved similarly with respect to M T B binding (P = 0.63 for comparison of binding at least one bacterium under non-opsonic conditions). The percentage of Day 0 A M from C57BL/6 or B A L B / c mice binding at least one bacterium under non-opsonic conditions were 15.3 + 3.2% and 13.0 + 3.0%, respectively (Figure 11). In the presence of serum, Day 0 C57BL/6 A M responded to the addition of serum with increased M T B binding whereas, similar to previous reports [18] Day 0 B A L B / c A M did not (Figure 11). The observed difference in M T B binding 54 between Day 0 C57BL/6 A M and Day 0 B A L B / c A M in the presence of serum was statistically significant and not dependent upon the mouse serum source (P = 0.002 and 0.001 for 1% N B M S and NMS, respectively). The serum-mediated enhanced binding of M T B by Day 0 C57BL/6 A M was seen using serum from either C57BL/6 or B A L B / c mice (Figure 11). The percentage of Day 0 C57BL/6 A M associated with at least one bacterium was 40.2 ± 3.3% and 42.0 ± 4.0% for 1% NBMS and 1% NMS, respectively (Figure 11). In contrast, neither C57BL/6 mouse serum nor B A L B / c mouse serum caused Day 0 B A L B / c A M to respond with increased M T B binding (Figure 11). The percentage of Day 0 B A L B / c A M associated with at least one bacterium was 15.5 + 4.6% and 15.2 + 2.3% for 1% NBMS and 1% NMS, respectively (Figure 11). The observed difference in M T B binding between Day 0 C57BL/6 A M and Day 0 B A L B / c A M emphasizes the role host factors may play in determining the outcome of an infection with M T B . 55 100 (-) 1%NBMS 1%NMS Figure 11. Comparison of M T B binding to Day 0 C57BL/6 A M and Day 0 B A L B / c A M in the presence and absence of serum. A M obtained from either CDI lb+/+ or B A L B / c mice were adhered to coverslips for 2 hours. The ability of these macrophages to bind M T B either in the absence of serum (-), presence of 1% B A L B / c mouse serum (1%NBMS) or 1% CDI lb+/+ mouse serum (1% NMS) was tested. The mean percentage + S E M of the macrophages binding at least one bacterium is reported for n = 4 (2 separate experiments with duplicate coverslips from each experiment). 56 3.7 Comparison of M T B Survival and Replication following Phagocytosis by either C D l l b - / - or CD11+/+ P M . To assess whether CR3-mediated entry provides M T B an advantage for intracellular survival and replication in macrophages, colony forming units (CFU) of M T B at 0, 1, 4, and 7 days following in vitro phagocytosis by either CDI lb-/- P M or CDI lb+/+ P M were determined. When P M of either phenotype were exposed to M T B at the same multiplicity of infection (MOI), the C F U of M T B at 7 days post-infection was significantly greater for CDI lb+/+ P M compared to CDI lb-/- P M (P = 0.031). However, based on the M T B binding data presented above, the higher C F U of M T B seen with CDI lb+/+ P M was possibly due to the increased efficiency with which CDI lb+/+ P M bind and internalize M T B . This claim is supported by comparing the C F U at Day 0 for the two macrophage populations infected with M T B at an MOI of 10. The C F U of M T B from CDI lb+/+ P M and CDI lb-/- P M at this point were 9.2 x 104 ± 1.0 x 103 and 6.3 x 104 ± 2.5 x 103, respectively (Figure 12 and Table 3), a significantly different uptake (P = 0.008). An estimated 4-5 population doublings occurred over the seven days for M T B that had been ingested by P M of either phenotype and the calculated doubling times for M T B from CDI lb+/+ P M and CDI lb-/- P M were 34.3 hours and 37.8 hours, respectively (Table 3). To control for the contribution of the initial number of M T B ingested, M T B survival and replication was also assessed for CDI lb-/- P M exposed to twice the MOI of M T B . This was designed to seed the same number of M T B in macrophages of either phenotype. Using an MOI of 20 for CDI lb-/- P M and an MOI of 10 for CDI lb+/+ P M resulted in the internalization of similar numbers of M T B , as measured by C F U on Day 0 (P = 0.143). 57 The corresponding C F U of M T B for CDI lb+/+ P M and CDI lb-/- P M on Day 0 were determined to be 9.2 x 104 + 1.0 x 103 and 1.0 x 105 + 5.0 x 103, respectively (Table 3). A comparison of the C F U at Days 1,4, and 7 following M T B phagocytosis demonstrated that, when similar numbers of M T B were internalized by either CDI lb-/- P M or CDI lb+/+ \"i P M , no significant difference in the C F U of M T B was observed (Figure 12 and Table 3; P 5e+6 o 4e+6 • 3e+6 -2e+6 -1e+6 -0e+0 -10CD11b+/+ 10CD11b-/-20CD11b-/ -0 1 4 7 D a y s P o s t I n f e c t i o n Figure 12. Comparison of M T B survival and replication following phagocytosis by either CDI lb-/- P M or C D I lb+/+ P M in vitro. P M obtained from either C D I lb-/- or C D I lb+/+ mice were adhered to coverslips overnight and exposed to M T B at the indicated MOIs for 3 hours in a serum-free environment. The CFU/ml of M T B for P M monolayers was then determined immediately (Day 0) or at 1,4, and 7 days post infection. The mean CFU/ml + S E M from two separate experiments with duplicate coverslips in each is shown. CFU/ml of M T B for CDI lb+/+ P M monolayers infected at a dose of 10 M T B per macrophage [10CD1 lb+/+] and CDI lb-/- P M monolayers infected at either 10 [10CD1 lb-/-] or 20 [20CDllb-/-] M T B per macrophage are shown. 58 Table 3. C F U and Calculated Population Doubling Time for M T B following Phagocytosis by either CDllb+/+ P M or C D l l b - / - P M in vitro CFU/ml at Number of Days Post Infection Mean (+ SEM) Doubling P M 0 1 4 7 Time (hours) 10CDllb+/+ 9.2 x 104 (1.0 x 103) 1.6 x 105 (6.5 x 103) 5.6 x 105 (2.1 x 104) 2.7 x 106 (2.1 x 105) 34.3 lOCDllb-/- 6.3 x 104 (2.5 x 103) 8.8 x 104 (6.5 x 103) 6.8 x 105 (1.4 x 104) 1.4 x 106 (1.4 x10 s) 37.8 20CDllb-/- 1.0 x 105 (5.0 xlO 3) 2.0 x 105 (1.7 x 104) 8.8 x 105 (1.6 x 105) 3.1 x 106 (7.5 x 105) 34.2 The CFU/ml and calculated population doubling times for MTB following phagocytosis by either CDI lb+/+ PM or CDI lb-/- PM in vitro are reported. PM from either CDI lb+/+ or CDI lb-/- mice were adhered to coverslips overnight and exposed to MTB at the indicated MOIs for 3 hours in a serum-free environment. The CFU/ml of MTB for PM monolayers was then determined immediately (Day 0) or at 1,4, and 7 days post infection. The mean CFU/ml + SEM from two separate experiments with duplicate coverslips in each is shown. CFU/ml of MTB for CDI lb+/+ PM monolayers infected at a dose of 10 MTB per macrophage [10CDllb+/+] and CDllb-/- PM monolayers infected at either 10 [lOCDllb-/-] or 20 [20CD1 lb-/-] MTB per macrophage are shown. The population doubling times for MTB were calculated using CFU values at Day 7 and Day 0 for each condition and the equation N t = N 0e k t which describes logarithmic growth rates where N t = number of bacteria at time = t, N 0 = number of bacteria at time = 0, k = growth rate constant, t = time and e = natural logarithm. 59 C H A P T E R 4 DISCUSSION: Role of CR3 in M T B Pathogenesis Investigated using C D l l b - / - and CDllb+/+ Mouse Macrophages The binding of M T B to host macrophages is the initial step in the establishment of infection. This important first step is the focus of much research based on the observation that other intracellular pathogens are able to survive inside macrophages following phagocytosis via some, but not all, receptor mediated pathways [60-63]. It has been speculated that M T B exploits CR3 to gain entry into macrophages without activating macrophage microbicidal functions because CR3-promoted phagocytosis can occur without triggering an oxidative burst [64,65]. In this study the contribution of CR3 to the binding of M T B by murine macrophages was examined using a CDI lb knockout mouse model and the role of complement in mediating the binding of M T B under conditions where serum is present was investigated. The hypothesis that M T B exploits CR3 as a means to favor its intracellular survival was also tested by comparing the growth of intracellular M T B up to 7 days following phagocytosis by either CDI lb-/- P M or CDl lb+/+PM. Particle binding experiments were used to characterize macrophage receptor function. These assays demonstrated that CDI lb-/- P M were less able to bind zymosan compared to their CDI lb+/+ counterparts (Figure 3). The difference in zymosan binding between CDI lb-/- P M and CDI lb+/+ P M is in agreement with recently reported observations and may be attributed to the lectin-like properties of CR3 [28]. No difference in the association of EIgG or E l g M C was detected between CDI lb-/- P M and CDI lb+/+ P M (Figure 3). 60 The conditions used to prepare E l g M C particles are designed to coat the erythrocytes with iC3b, the principal ligand for CR3, therefore it is surprising to find that CDI lb-/- P M associated with E l g M C at levels comparable to CDI lb+/+ P M . It is possible that P M from the CDI lb knockout mouse have compensated for the absence of CDI lb by expressing higher than normal amounts of other receptors with common binding specificity. Presumably, if this were the case the CDI lb knockout macrophages would express greater amounts of CR4 or CR1 because these receptors have binding functions in common with CR3. However, the flow cytometry data do not support the idea that the CDI lb knockout mouse compensated for the loss of CR3 by expressing greater amounts of CR4, CR1, or any of several other receptors that were measured (Figure 2). It is also possible that normal levels of CR1 and/or CR4 present on CDI lb-/- P M take over the task of binding E l g M C . The latter could be tested using competitive ligands, such as anti-CRl and anti-CR4 antibodies, for the complement binding sites on CR1 and CR4. Although the protocol used to generate E l g M C was designed to favor the deposition of iC3b, E l g M C may also have C3b on their surface and therefore comparable levels of E l g M C binding between CDI lb+/+ P M and CDI lb-/- P M may also be due to CR1-mediated binding to C3b deposited on E l g M C . The association of M T B with CDI lb-/- P M in the absence of serum was significantly less than that observed for CDI lb+/+ P M (P = 0.008). Approximately 20% fewer macrophages bound M T B in CDI lb-/- monolayers compared to CDI lb+/+ monolayers (Figure 4). This observation indicates that CR3 is a quantitatively important receptor for M T B under serum-free conditions but that CR3 does not account for the majority of binding. These observations also indirectly support the hypothesis that other macrophage 61 receptors are important for the non-opsonic binding of M T B . Alternate candidate receptors for the non-opsonic binding of M T B include MR, CD14, and CR4 [8,14,32]. Dissecting the relative contribution of each of these receptors in mediating the interaction of M T B with macrophages and the outcomes of these interactions will be critically important in increasing our understanding of tuberculosis. Although CR3 was shown to play a limited role in the non-opsonic binding of M T B , a dramatic difference in M T B binding was observed between CDI lb-/- P M and CDI lb+/+ P M in the presence of serum. At the greater than 30 bacteria per macrophage level, CDI lb-/- P M associated with M T B approximately 9x less efficiently than did CDI lb+/+ P M (Figure 5). This observation confirms the importance of CR3 for the efficient uptake of opsonised M T B and suggests that CR3 is the predominant receptor for M T B in the presence of serum. Presumably, the enhanced binding of M T B to CR3 in the presence of serum is mediated by the interaction of opsonic iC3b that has been deposited on M T B and the iC3b-binding site of CR3. Whether CR3 functions as the primary receptor or cooperates with other receptors to mediate the uptake of M T B was not investigated. However, recently, it has been proposed that CR3 cooperates with other macrophage receptors including Fc receptors and C D 14 to mediate the internalization of bound particles [33]. One plausible model suggests that alternate receptors, such as CD 14, serve as scouts for CR3, thus expanding the ligand binding repertoire of CR3 [33]. Because CD 14 has been reported to bind L A M of M T B [11,32] this model has interesting implications for the role of CR3 in the pathogenesis of 62 tuberculosis. Future experiments using knockout mice with deficiencies in multiple receptors could be used to investigate receptor cooperation in M T B phagocytosis. The binding of M T B in the presence of serum was further investigated using heat-inactivated serum (HI) and R A G serum. The latter is obtained from RAG-1 (-/-) mice and does not contain immunoglobulins [51] whereas HI does not have the capacity for complement activation [53]. When HI was used as a source of opsonins, the association of M T B with P M of either phenotype returned to levels similar to non-opsonic association (Figure 6), suggesting that, for both CDI lb+/+ and CDI lb-/- P M , complement activation was responsible for the enhanced binding of M T B . Binding studies with R A G serum demonstrated that immunoglobulins were not important for the enhanced binding of M T B seen in the presence of NMS. Contrary to claims made by Hetland et al. [55], the observations made here indicate that immunoglobulin-mediated classical complement pathway activation is not important for the enhanced uptake of M T B by macrophages. Due to recent developments and controversy concerning the role of complement in mediating the enhanced binding of M T B to macrophages I was interested in characterizing the role of either the classical or alternative complement pathways in mediating M T B binding. Using either factor B-depleted or C2-depleted human serum to evaluate, respectively, the contribution of the alternative or classical complement pathways, I found that with CDI lb+/+ P M the majority of M T B binding was enhanced via activation of the classical and not the alternative complement pathway. C2-depletion greatly reduced the enhanced binding of M T B and brought binding to levels comparable to those seen when 63 serum was absent. There was approximately a nine-fold decrease in the percentage of macrophages associated with greater than 30 bacteria when C2-depleted serum was used as a source of opsonins instead of PHS (Table 2). On the other hand, the decrease in M T B binding seen with factor B-depleted serum was approximately two-fold at the greater than 30 bacteria per macrophage level (Table 2). The experiments described here do not support the claim that enhanced binding of M T B by macrophages is mediated primarily through activation of the alternative pathway [6] or by the immunoglobulin-mediated classical pathway [54]. A possible explanation for the disagreement with these earlier reports is that Schlesinger et al. examined the role of complement in M T B binding using factor B-depleted serum but did not assess the effects of C2-depleted serum on M T B binding [6]. Whereas, Hetland et al. draw their conclusions from experiments using chelating agents and serum from tuberculoid and non-tuberculoid individuals to determine whether B C G activates either the classical or alternative pathways of complement [54]. I studied the effects of E D T A and E G T A on the binding of M T B because these chelating agents have been widely used as a means to investigate the contribution of either the classical or alternative pathways [6, 54]. At the concentration used to inactivate complement, EDTA and E G T A have an effect on the binding of M T B to macrophages beyond complement inactivation. Indeed, the cationic-dependent function of several macrophage receptors, including CR3, has been documented [66,67]. Therefore, in light of the observations made here, previous work using E D T A or E G T A as a means to investigate the opsonic binding of pathogens by macrophages should be revisited and tested with the appropriate factor depleted sera. 64 Taken together, the observations with R A G serum and C2-depleted serum indicate that, in the presence of normal serum, enhanced binding of M T B by macrophages is mediated predominantly via activation of the classical complement pathway and occurs independently of immunoglobulins. The immunoglobulin-independent enhanced binding of M T B by macrophages in the presence of serum may be attributed to the complement activating potential of the mannose-binding protein (MBP) [68]. M B P , a lectin specific for mannose and iV-acetylglucosamine, has structural homology with C l q and reportedly activates the classical complement pathway [68]. Therefore, it is conceivable that binding of M B P to mannose residues on M T B triggers the deposition of opsonic C3 peptides on the M T B surface, in a C2-dependent manner, which leads to enhanced binding of M T B by macrophages. In addition, the observation that M T B has developed an invasion strategy which is based on the acquisition of opsonic C3 peptides by scavenging C2a to create a novel C3 convertase [16] could also explain some of the results presented here. Contrary to CDI lb+/+ P M , the role of either the classical or alternative pathways in mediating enhanced binding of M T B appears to be equivalent with CDI lb-/- P M . When either factor B-depleted or C2 depleted serum were used there was a significant decrease in the binding of M T B compared to PHS (Table 2). At the present time, no definitive explanation for the observed difference in complement activation between CDI lb-/- P M and CDI lb+/+ P M can be presented. However, the observation that CDI lb-/- P M behave differently than CDI lb+/+ P M with respect to complement-mediated M T B binding supports the hypothesis that CR3 is an important receptor in mediating the opsonic association of M T B with macrophages. 65 Previously, Stokes et al. reported that the phenotype of a macrophage greatly affects its interaction with mycobacteria and demonstrated that P M , although widely cited in the literature as a model to investigate mycobacteria-macrophage interactions, do not behave like A M with respect to M T B binding [18]. In the current studies, the role of CR3 in M T B binding was initially assessed with experiments using P M . There are several reasons why P M were used initially, including the feasibility of their isolation and the fact that P M are widely cited in the literature as models for investigating macrophage properties. However, I was also interested in characterizing the role of CR3 in M T B binding using A M because murine A M closely resemble the resident human alveolar macrophage phenotype [18] and A M are the initial host cell in clinical M T B infections. Particle binding assays failed to demonstrate a difference in the binding of any of the control particles between A M of either phenotype. Unlike P M , no difference in zymosan binding was observed between CDI lb-/- A M and CDI lb+/+ A M (Figure 7). Compared to P M , A M do not express high levels of CR3 [12, 18], therefore the low expression of CR3 on A M may not allow for the detection of the contribution of CR3 to binding zymosan. However, A M associated with zymosan very efficiently and presumably the binding of zymosan by A M is mediated by other receptors such as the MR. Like P M , no significant difference in EIgG or E l g M C binding was detected between CDI lb-/- A M and CDI lb+/+ A M . Again the absence of a detectable difference between macrophages of either phenotype to associate with E l g M C was unexpected. However, it has been shown previously that A M do not associate with E l g M C very efficiently [18] and therefore, it is 66 also possible that the low expression of CR3 on A M does not allow one to detect differences in the association of E l g M C between CDI lb-/- and CDI lb+/+ A M . The pathophysiologic relevance of M T B infection in the lung makes characterizing the non-opsonic interaction of M T B with host macrophages an important area of research. Comparable with the observations that were made with P M , CDI lb-/- A M were less able to associate with M T B compared to their CDI lb+/+ counterparts. Of particular interest was the observation that the absence of CDI lb on A M depressed M T B binding modestly under non-opsonic conditions. This observation is in disagreement with a previous report by Cywes et al. which suggested that CR3 is the major receptor for M T B under non-opsonic conditions [13]. In fact, the observations made here suggest that the majority of M T B binding to A M occurs independently of CR3, thereby providing support for the claim that alternate receptors such as CR4 or M R are responsible for mediating the binding of M T B under non-opsonic conditions [14,69]. The role of CR3 in mediating the efficient uptake of M T B in the presence of serum was also demonstrated for A M . CDI lb-/- A M associated with M T B less efficiently than CDI lb+/+ A M in the presence of serum. Examining the effects of serum on the association of M T B with A M demonstrated that, similar to our observations with P M , the enhanced binding of M T B under opsonic conditions is mediated by a heat-labile serum component(s) but is independent of serum immunoglobulins. This observation indirectly supports the hypothesis that enhanced binding of M T B by macrophages is mediated by immunoglobulin-independent complement activation. However, the role of the classical 67 versus the alternative pathway for complement activation and C3b fixation in mediating the binding of M T B to A M was not examined because previous work has shown that mouse A M do not respond to human serum with increased binding of M T B (unpublished observations Stokes laboratory). This factor, in conjunction with the unavailability of C2-depleted or factor B- depleted mouse sera made the relevant experiments difficult to perform. The apparent serum incompatibility of murine A M but not murine P M with human serum cannot yet be readily explained. It is possible that differences in the expression and/or specificity of CRs on murine A M compared to P M do not permit efficient complement activation when human serum is used as a source of opsonins. For example, mouse CRI does not recognize human complement proteins [70-72]. Therefore, if CRI on mouse A M is important for the serum-mediated binding of M T B , the serum incompatibility may explain the requirement for mouse serum as a source of opsonins for M T B binding by mouse A M . An alternative explanation is that M T B binding to A M in the presence of serum is not predominantly complement mediated and that the heat sensitive serum factor(s) responsible for the enhanced binding of M T B by A M is present in mouse serum but not present in human serum. Previous reports have suggested that the increased expression of surface receptors, including CR3, which accompanies differentiation of A M are responsible for increased binding of M T B [18]. Stokes et al. previously reported that Day 0 A M bind M T B poorly and that M T B binding is not enhanced by the addition of serum to the binding medium [18]. However, after four days of differentiation in vitro, A M respond to the addition of serum with enhanced binding of M T B [18]. In the present study, I was interested in testing 68 whether the enhanced binding seen by Day 4 A M in the presence of serum was due to the increased expression of CR3. Based on the observations, it appears that a portion of the enhanced binding could be attributed to CR3 but that the majority of M T B binding by either Day 0 or Day 4 A M was not mediated by CR3. In addition, I also found that murine Day 0 A M from different strains of mice do not behave uniformly with respect to M T B binding. Day 0 A M obtained from either CDI lb-/- or CDI lb+/+ mice, both of which are of similar genetic background as C57BL/6 mice, responded to the addition of serum with enhanced M T B binding. Whereas, similar to previous reports, Day 0 A M from B A L B / c mice did not respond to the addition of serum with enhanced M T B binding [18]. The difference in M T B binding seen between Day 0 A M from B A L B / c mice and C57BL/6 mice was due to functional differences in the A M themselves as these differences were observed when serum from either mouse strain was used as a source of opsonins. Further experiments would be required to provide a clear explanation for the observed difference in M T B binding between Day 0 A M from C57BL/6 and B A L B / c mice. However, it is possible that the increased binding of M T B , in the presence of serum, by Day 0 A M from C57BL/6 compared to Day 0 A M B A L B / c was due to differences in receptor expression. The latter could be examined using flow cytometric analysis and presumably Day 0 A M from C57BL/6 mice would express greater amounts of receptors which mediate the binding of M T B in the presence of serum. The M T B survival and replication experiments performed here assessed the role of CR3 in determining the intracellular viability of M T B following phagocytosis under non-opsonic conditions. Non-opsonic conditions were used for infecting macrophages in an attempt to 69 mimic the initial interaction of M T B with human A M . Admittedly, the use of P M is less desirable than A M . However, based on the previous observations that A M do not express high levels of CR3 whereas P M do express high levels of CR3,1 felt that for these experiments P M would provide a better means to assess the role of CR3. In testing whether the absence of CR3 affects the intracellular fate of M T B , no significant difference was observed between CDI lb-/- P M and CDI lb+/+ P M in the survival and replication of M T B following phagocytosis of similar numbers of M T B (P > 0.05 for all comparisons). Therefore the observations made here do not support the hypothesis that CR3-mediated entry provides M T B with an advantage for intracellular survival and replication. It would be interesting to see if the same conclusion would be made following phagocytosis under opsonic conditions. It has been reported previously that binding to the iC3b-binding site on CR3 leads to phagocytosis without an accompanying oxidative burst [36] whereas, engagement of both the 0-glucan binding site and the iC3b binding site on CR3 is accompanied by an oxidative burst [22,23,28]. Therefore, it is possible that CR3 alters the intracellular fate of M T B but only does so under conditions where serum is present. The latter could be tested by modifying the survival and replication experiments described here to compare the viability of M T B following phagocytosis in the presence of serum. 70 C H A P T E R 5 R E S U L T S : Interaction of M T B with M H - S Cells Compared to Primary Macrophages 5.1 Comparison of M T B binding to M H - S cells, A M and P M . Similar to previously reported findings [12], the observations made here demonstrated that the binding of M T B to different macrophage phenotypes varied. Under non-opsonic conditions, 79.0 + 12% of resident B A L B / c P M were associated with at least one bacterium compared to only 36.0 ± 8 . 1 % of B A L B / c A M (Figure 13; P < 0.001). Of significant interest to the overall aim of testing whether the MH-S cell line was a suitable model for investigating macrophage-MTB interactions was the observation that MH-S cells behaved similarly to their in vivo counterpart, the mouse A M , with respect to M T B binding under non-opsonic conditions. Under the conditions tested, 35.7 + 12.7% of M H -S monolayer population bound at least one bacterium (Figure 13). No significant difference was observed among the three macrophage populations in their ability to bind latex particles (Figure 13; P > 0.3 for comparisons of all three macrophage cell types). This demonstrated that the decreased binding of M T B by MH-S cells and A M was not due to a global inability of these two cell populations to bind particles. 71 MTB Latex Figure 13. Binding of M T B and Latex Beads to P M , A M , and MH-S cells. P M obtained from BALB/c mice and MH-S cells were adhered to coverslips in serum-supplemented medium for 18 hours. A M obtained from BALB/c mice were adhered to coverslips and cultured for 4 days. The ability of these macrophages to bind M T B or 0.8 um polyvinyl latex beads (Latex) in a serum-free environment was then assessed. The percentage of the macrophage population binding > 1 M T B or > 1 latex bead is shown. The mean + S E M is shown for n = 12 (6 experiments with duplicate coverslips from each experiment). 7 2 5.2 M H - S , A M , and P M binding of EIgG, E l g M C and zymosan particles. Binding of control particles was used to probe for the expression of functional receptors for IgG (EIgG), complement component iC3b (E lgMC) , and glycans (zymosan) on the surface of the three macrophage populations. Under the conditions used for these experiments, P M did not bind E l g M C as efficiently as zymosan or EIgG (Figure 14) with approximately 40% of population binding E l g M C compared to nearly 100% of the population binding EIgG or zymosan. A M bound zymosan particles very efficiently with approximately 100% of A M ingesting an average of 20-25 zymosan particles (Figure 14). The association of EIgG with A M was moderate whereas the association of E l g M C with A M was negligible (Figure 14). MH-S cells were observed to bind EIgG and zymosan moderately but the binding of E l g M C by MH-S cells was also negligible (Figure 14). The data demonstrate that, compared with A M , MH-S cells exhibit similar binding properties with respect to EIgG and E l g M C but bind zymosan less efficiently (P for EIgG and E l g M C association = 0.13 and 0.47, respectively and P < 0.001 for zymosan association). Compared to P M , MH-S cells exhibit an impaired ability to bind all three particles tested, however, the difference in EIgG binding was not significant (P > 0.05 for EIgG and P = 0.007 and 0.008 for zymosan and E l g M C , respectively). 73 2500 Zymosan EIgG ElgMC Figure 14. Binding of Zymosan, EIgG, and E lgMC' to P M , A M , and MH-S cells. P M obtained from B A L B / c mice and MH-S cells were adhered to coverslips in serum-supplemented medium for 18 hours. A M from B A L B / c mice were adhered to coverslips and cultured for 4 days. The ability of these macrophages to bind Zymosan, EIgG, and E l g M C in a serum-free environment for 3 hours was tested. Binding was assessed as the number of particles associated with 100 macrophages (Association Index). The mean + S E M is shown for n = 6 (3 separate experiments with duplicate coverslips in each experiment). Note: the binding of E l g M C by MH-S was negligible with an association index of4.7 + 5.0. 74 5.3 M H - S cells, A M and P M binding of M T B in the presence of serum. Because MH-S cells behaved similarly to A M with respect to M T B binding in a serum-free environment I was interested in characterizing M T B binding by MH-S cells in the presence of serum. With increasing serum concentrations of 0%, 1%, and 5%, the percentage of A M binding M T B were 36 ± 2.6%, 66.7 ± 8.3%, and 83 ± 4.3%, respectively (Figure 15). In the presence of serum, an increase in the average number of M T B per A M was also observed. Approximately 40% of the A M contained greater than 10 bacteria in the presence of 5% serum compared to less than 1% when no serum was present (Figure 15). The percentage of MH-S cells binding M T B also increased significantly in the presence of serum (P = 0.041), but the increased binding of M T B was less than that seen with A M . With increasing serum concentrations of 0%, 1%, and 5% the percentage of MH-S binding M T B were 40.3 ±3.0%, 49.2 ± 3.9%, and 52.5 ± 3.4%, respectively (Figure 15). Unlike A M , the increase in the percentage of MH-S cells binding M T B in the presence of serum did not include the majority of cells. Instead, a subpopulation of MH-S cells responded to the addition of serum with increased binding of M T B . In the presence of 5% N B M S , approximately 20% of MH-S cells associated with greater than 10 bacteria compared to less than 2% when no serum was present (Figure 15). Neither MH-S nor A M were able to associate with M T B to levels seen with P M . As previously reported, the binding of M T B by P M was dramatically enhanced by the addition of serum [12]. With increasing concentrations of serum of 0%, 1%, and 5% the percentage of P M binding M T B were 66.7 ± 1.6%, 85.5 ± 3.0%, and 96.5 ± 0.7%, respectively (Figure 15). The enhancement of M T B binding in the presence of serum also corresponded with a marked increase in the average number of bacterium per macrophage. 75 In the presence of 5% normal mouse serum, approximately 90% of the P M contained greater than 10 bacterium compared to less than 30% of cells binding greater than 10 bacterium when no serum was present (Figure 15). 76 100 • 80 • 60 • 40 20 CQ • 0 1— 100 T 80 c 60 Bindi 40 20 0 100 80 60 40 20 MH-S > 1 bacter ium w — > 10 bacter ia (-) 1 % N B M S 5 % N B M S Figure 15. Binding of M T B by P M , A M , and MH-S cells in the presence of normal B A L B / c mouse serum (NBMS). P M obtained from B A L B / c mice and MH-S cells were adhered to coverslips in serum-supplemented medium for 18 hours. A M obtained from B A L B / c mice were adhered to coverslips and cultured for 4 days. The binding of M T B was assessed in the absence of serum (-) or presence of 1% or 5% N B M S . The mean percentage of the macrophages binding either at least 1 or greater than 10 M T B is reported for n = 12 (6 separate experiments with duplicate coverslips from each experiment). 77 5.4 Effect of serum heat-inactivation on the association of M T B with M H - S cells The enhanced binding of M T B by macrophages seen in these experiments could be attributed to several different macrophage receptors. However, it is generally accepted that complement receptors mediate this enhanced binding because heat treatment of the serum abrogates the enhancement [6,18]. Therefore to test whether complement activation was responsible for the enhanced binding of M T B by MH-S cells I compared the binding of M T B by MH-S cells in the presence of N B M S and HIBMS. In this series of experiments the percentage of MH-S cells associated with at least one bacterium was 56.8 + 5.6% and 55.4 + 4.0% for 1% and 5% N B M S , respectively (Figure 16). When HIBMS was used as a source of opsonins, the percentage of MH-S associated with at least one bacterium dropped to 25.2 ± 4.3% and 33.2 + 3.0% for 1% HIBMS and 5% HIBMS, respectively (Figure 16). The association of M T B with MH-S in the presence of HIBMS was comparable to that seen under non-opsonic conditions, with 34.2 + 8.7% of MH-S associated with at least one bacterium in the absence of serum (Figure 16). In addition, when HIBMS was used as a source of opsonins, less than 0.5% of MH-S cells associated with greater than 10 bacteria (Figure 16). Therefore the observations made here indicate that the serum-mediated enhanced binding of M T B by MH-S is mediated by a heat-labile serum component and suggests that, similar to primary macrophages, M T B binding was enhanced by opsonization with complement proteins. 78 100 Figure 16. Effect of serum heat inactivation on the binding of M T B by MH-S cells. MH-S cells were adhered to coverslips in serum-supplemented medium for 18 h. The binding of M T B in the absence of serum or presence of 1% N B M S , 1% heat-inactivated B A L B / c mouse serum (HIBMS), 5% N B M S , or 5% HIBMS was assessed. The mean percentage of MH-S cells binding either at least 1 or greater than 10 M T B is reported for n = 6 (3 separate experiments with duplicate coverslips from each experiment). 79 5.5 Role of CR3 in the association of M T B with MH-S cells Based on the observation that approximately 20% of MH-S cells responded to the addition of serum with increased binding of M T B , I hypothesized that this subpopulation of cells represented a CR3-positive population of MH-S. To test this hypothesis, I assessed the effects of two different anti-CR3 monoclonal antibodies on the enhanced binding of M T B by MH-S. Compared with a non-specific rat IgG control (IR863) antibody, both Ml /70 and 5C6, two different anti-CR3 monoclonal antibodies recognizing different epitopes on CR3, inhibited the binding of M T B by MH-S significantly (P = 0.016 and 0.023 for M l / 7 0 and 5C6, respectively). The percentage of MH-S cells associated with one or more M T B was reduced from approximately 40% to 17.2 + 5.3% and 16.0 + 6.8% in the presence of Ml /70 and 5C6, respectively (Figure 17). 80 100 Figure 17. Role of complement receptor type 3 (CR3) in binding of M T B by MH-S cells in the presence of N B M S . MH-S cells were adhered to coverslips in serum-supplemented medium for 18 hours. The effects of anti-CR3 monoclonal antibodies on the binding of M T B in presence of 1% N B M S with non-specific rat IgG (Control) or with anti-CR3 monoclonal antibodies (Ml/70 and 5C6) was tested. The mean percentage of MH-S cells binding at least 1 or greater than 10 M T B is reported for n = 6 (3 separate experiments with duplicate coverslips from each experiment). 81 5.6 Isolation of CR3-negative population of M H - S cells Binding studies with anti-CR3 antibodies indicated that CR3 played a major role in mediating the binding of M T B by MH-S cells under opsonic conditions. However, because binding assays using antibodies as competitive inhibitors cannot be considered definitive, I wanted to dissect the role of CR3 in mediating M T B binding using CR3-negative and CR3-positve populations of MH-S cells. In the original publication describing MH-S cells the authors report that approximately 10% of MH-S cells are Mac-1 positive and that CR3-negative and CR3-positive subpopulations of MH-S cells were isolated using a cell sorter [42]. Based on this information, I attempted to isolate CR3-negative and CR3-positive subpopulations of MH-S cells using immunoselection. Using this method as a means to isolate receptor negative cell populations, one would expect to see an increase in the proportion of CR3-negative cells with each successive round of immunoselection. However, the ratio of CR3-negative to CR3-positive cells for rounds one, two, and three of immunoselection were 4.9:1, 6.1:1, and 2.6:1, respectively (Figure 18). Thus, contrary to the previous report [42], I was unable to isolate stable CR3-positive and CR3-negative subpopulations of MH-S cells and based on the observations made over several separate trials it appears that the expression of CR3 on MH-S cells is transient or at the very least fluctuates within a given MH-S population. 82 Round 1 Round 2 Round 3 Figure 18. Isolation of CR3-negative subpopulations of MH-S cells. MH-S cells were exposed to a cocktail of rat anti-mouse CR3 monoclonal antibodies and MH-S cells which bound anti-CR3 monoclonal antibodies were magnetized by exposing the cells to iron beads coated with sheep anti-rat immunoglobulins. CR3-expressing cells were then removed from the cell suspension using a magnetic apparatus. The resulting CR3-negative MH-S cells were then cultured for 24-48 hours and representatives of this population were again subjected to the immunoselectiOn procedure. The percentage of CR3-negative [CR3(-)] and CR3-positive [CR3(+)] cells is reported for three successive rounds of immunoselection. 83 C H A P T E R 6 DISCUSSION: Interaction of M T B with M H - S Cells Compared to Primary Macrophages The binding properties of MH-S cells, an immortalized alveolar macrophage cell line, were compared with primary murine macrophages to determine whether the MH-S cell was a suitable model for investigating macrophage-MTB interactions. The ultimate objective of these experiments was to use CR3-negative and CR3-positive populations of MH-S cells as a means to dissect the role of CR3 in mediating the binding of M T B with macrophages. Using an immortalized cell line as a means to investigate the contribution of CR3 in mediating the binding of M T B by macrophages was also seen to be advantageous from a cost perspective as well as reducing the use of animals for experimental purposes. Characterizing the non-opsonic binding of M T B by MH-S cells demonstrated that the association of M T B with MH-S was similar to that seen with A M . In the absence of serum, the distribution in the binding of M T B by MH-S was also similar to A M , in that less than 1% of the cells in the monolayer bound greater than 10 bacteria (Figure 14). Neither MH-S nor A M bound M T B as well as P M (Figure 12). It has been shown previously that the binding of M T B either opsonically or non-opsonically to P M is significantly greater than that seen with A M [18]. P M and A M were used in these experiments as a means to compare the binding properties of MH-S cells to other macrophage models and as examples of how M T B binding differs among different macrophage phenotypes. 84 Particle binding experiments demonstrated that MH-S cells behave similarly to A M with respect to EIgG and E l g M C ' binding (Figure 13). However, one striking difference in the binding properties of MH-S cells compared to A M was the reduced ability to bind zymosan by MH-S. Compared to A M the binding of zymosan by MH-S was reduced approximately 6-fold, with association indices of 2334 + 174 and 392 + 11, respectively (Figure 13). Recently, CR3 has been reported to be an important receptor for unopsonized zymosan and the binding of zymosan by CR3 is believed to occur at the CR3 glycan binding site [28]. However, several observations suggest that the majority of zymosan binding by macrophages occurs independently of CR3. First, flow cytometry data indicate that A M and MH-S have similar CR3 expression [42] but A M associated with zymosan much more efficiently than MH-S (Figure 14). Second, E l g M C association assays demonstrated that CR3 function on A M was poor but this did not correlate with poor zymosan binding (Figure 14). Third, P M associated with zymosan less efficiently than A M but CR3 function, measured here by E l g M C binding (Figure 14), and CR3 expression, measured by flow cytometric analysis and reported previously [12,18], indicates that CR3 expression and function is greater for P M than A M . The observations made here support the claim that the binding of zymosan by A M reflects the role of lectin-like receptors, other than CR3, on macrophages in the normal immuno-surveillance of lungs in vivo [18]. The binding of fungal pathogens by A M has been documented [73,74] and presumably the mechanism for binding fungal pathogens is common to zymosan binding. A possible explanation for the inferior binding of zymosan 85 by MH-S cells is that due to continuous culturing in vitro the MH-S cell line has lost the physiological response to stimuli that drive the expression of receptors for zymosan. Investigating the association of M T B with MH-S cells under opsonic conditions demonstrated that, similar to P M and A M , the enhanced binding of M T B by MH-S cells was due to complement activation since mild heat treatment of serum abrogated the enhanced binding (Figure 16). However, contrary to observations with P M and A M , MH-S cells did not respond uniformly to the addition of serum. Over several separate experiments the percentage of MH-S cells that responded to the addition of serum with increased M T B binding ranged from 10-25% (Figure 15 and 16). It is possible that these serum-responsive cells represent a CR3-positive subpopulation of MH-S. This claim would be consistent with the original report on the phenotypic characteristics of MH-S, where the authors report that approximately 10% of the MH-S parental strain are Mac-1 positive [42]. Indeed, previous studies have identified the quantitatively important role of CR3 in mediating MTB-macrophage interactions [12,13]. Therefore, I considered the possibility that a subpopulation of CR3-expressing MH-S cells was responsible for the increased binding of M T B . In binding assays using two different anti-CR3 monoclonal antibodies, Ifound that M T B binding by MH-S cells was blocked significantly (P < 0.02 for either antibody). An approximate 2-fold reduction in the percentage of MH-S cells associated with M T B was observed in the presence of either Ml /70 or 5C6 (Figure 17). Presumably the inhibition caused by 5C6, the non-iC3b site binding monoclonal antibody, was due to steric effects on CR3 whereas Ml /70 inhibition was due to competitive inhibition with the iC3b-binding site on CR3. 86 Having demonstrated that anti-CR3 monoclonal antibodies could dramatically reduce the binding of M T B by MH-S cells, I was interested in characterizing further the role of CR3 in mediating the binding of M T B . Specifically, I wanted to isolate CR3-negative and CR3-positive populations in the MH-S cell line and compare the binding properties of these two different MH-S subpopulations. However, after several rounds of immunoselection and numerous attempts I was unable to isolate stable CR3-negative populations of MH-S cells. In fact, contrary to previous reports [42] the observations made here indicate that CR3 was transiently expressed on MH-S cells as successive rounds of immunoselection failed to enrich the cell population for CR3 negative cells (Figure 17). Based on the latter observation, I did not pursue using the MH-S cell line as a model to assess the role of CR3 in mediating the interaction of M T B with macrophages. 87 C H A P T E R I C O N C L U S I O N Collectively, the evidence presented here indicates that CR3 is not essential for the binding of M T B either opsonically or non-opsonically by murine macrophages in vitro. Under opsonic conditions, CR3 was required for the efficient uptake of M T B by macrophages. However, the absence of CR3 did not alter the intracellular viability of M T B that had been internalized under serum-free conditions. Although the experiments performed here did not examine the role of CR3 in vivo, given the numerous functions of CR3, including the recruitment of monocytes and neutrophils to sites of inflammation and the observation that CR3 is required for the efficient uptake of M T B in the presence of serum, it is possible that CR3 plays an important role in pathogenesis of tuberculosis. Evidence for the latter would require performing in vivo experiments comparing the pathological hallmarks of M T B in CDI lb-/- and CDI lb+/+ animal models. Future experiments using the CDI lb-/- mouse model should facilitate a fuller understanding of the role of CR3 in the pathogenesis of tuberculosis. In addition, experiments using animal models with deficiencies in other receptors should clarify whether alternate receptors are required for the association of M T B with macrophages and whether or not other receptors are exploited by M T B as a means to avoid macrophage microbicidal mechanisms. The work with the MH-S cell line did not rule out the potential of MH-S cells to be used as a model for investigating M T B pathogenesis. However, based on the observations presented here, caution should be exercised when using MH-S cells to characterize the receptor-ligand interactions between M T B and macrophages. 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Unbound serum antigens were washed three times with wash buffer (PBS + 0.05% Tween 20) and then blocked with 1% milk powder in wash buffer for 30 minutes at room temperature. Alkaline-phosphatase conjugated goat anti-mouse IgG secondary was then added (1/1000 with wash buffer) to each well and incubated for 2 hours at room temperature. After this incubation step, wells were washed 3 times with wash buffer and alkaline-phosphatase substrate para-Nitrophenyl phosphate (Sigma Product No. 9389) was added. Absorbance (A405nm) readings were then made each 30 minutes up to 1.5 hours. The A405nm readings reported above are representative of the averages of duplicate wells from one ELISA. 97 APPENDIX II: Sample Calculation of MTB Population Doubling Time where N t = number of bacteria at time = t N 0 = number of bacteria at time = 0 k = growth rate constant t = time To find k, use Eq. l and values for N(7) and No Sample calculation for 10CD1 lb+/+ M T B data: 2.74e6 = 9.2e4ek(7) k = 0.4848 day 1 To calculate for t; one population doubling implies Nt = 2No and therefore substituting 2No for Nt gives equation 2. Eq.2 2No = Noekt Also have 2 = e k t and therefore, t = ln2 / k The doubling time for M T B from 10CD1 lb+/+ is calculated as follows: t = ln2/0.4848 day-1 = 1.4296 days or 34.3 hours Similarly, it is possible to calculate the population doubling times for M T B from the 10CD1 lb-/- and 20CD1 lb-/- monolayers. The calculated population doubling times for M T B from lOCDl lb - / - and 20CDllb- / - monolayers were 37.8 hours and 34.2 hours, respectively. Eq. 1 N t = N0e' 98 "@en ; edm:hasType "Thesis/Dissertation"@en ; vivo:dateIssued "1999-11"@en ; edm:isShownAt "10.14288/1.0089150"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Pathology"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Role of complement receptor type 3 in mycobacterium tuberculosis pathogenesis"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/9724"@en .