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Originally published online as doi:10.1189/jlb.1005582 on September 22, 2006

Published online before print September 22, 2006
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(Journal of Leukocyte Biology. 2007;81:212-220.)
© 2007 by Society for Leukocyte Biology

Mycobacteria-primed macrophages and dendritic cells induce an up-regulation of complement C5a anaphylatoxin receptor (CD88) in CD3+ murine T cells

Mary Anne Connelly*, Rachel A. Moulton*, Amanda K. Smith*, Devin R. Lindsey*, Meenal Sinha{dagger}, Rick A. Wetsel{dagger} and Chinnaswamy Jagannath*,1

* Department of Pathology and Laboratory Medicine, University of Texas Health Sciences Center, Houston, Texas, USA; and
{dagger} Institute of Molecular Medicine, Houston, Texas, USA

1Correspondence: Dept. of Pathology and Laboratory Medicine, University of Texas Health Sciences Center, MSB 2.200, 6431 Fannin, Houston, TX 77030, USA. E-mail: chinnaswamy.jagannath{at}uth.tmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Complement C5a anaphylatoxin is a potent activator of macrophages, neutrophils, and dendritic cells (DC) and binds the C5a receptor (C5a-R; CD88). Although C5a is chemotactic for T cells, expression of C5a-R on murine T cells has been disputed. We report here that naïve, Con A-activated, and cytokine (IL-12, IL-18)-stimulated murine CD3+ T cells from three strains of mice [C57Bl/6, B10.nSn (C5+/+), B10.on (C5–/–)] lacked C5a-R, as evaluated by immunophenotyping with an anti-C5a-R mAb. Ligation of CD3 induced a modest up-regulation with 3% of CD3+ T cells expressing cell surface C5a-R. T cells primed by APC differentiate into effector T cells. Activation of mycobacteria [bacillus Calmette-Guerin (BCG)]-sensitized T cells through MHC II and TCR interactions via BCG-infected macrophages enhanced the expression of C5a-R with ~14% of CD3+ T cells positive for C5a-R. Comparable expression was found in C5+/+ as well as C5–/– strains of mice (14% and 15%, respectively). Furthermore, anti-CD3-activated T cells were primed by BCG-infected DC, and a larger proportion of the primed T cells expressed C5a-R (30–40%). Finally, mice infected with BCG showed significant numbers of CD3+ T cells expressing C5a-R in the spleens during infection. As APC, such as macrophages and DC, can secrete C5 and cleave C5 to C5a and C5b through a peptidase, we suggest that macrophage and DC-T cell interactions can up-regulate C5a-R on T cells through MHC II-TCR and provide a C5a peptide for additional local activation of T cells via C5a-R.

Key Words: macrophages • BCG • mouse


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Complement components have multiple and varied effects on biological events in mammals [1 ]. The most potent anaphylatoxin of the complement system is C5a, the 14-kDa cleavage product of C5. C5a binds to the C5a receptor (C5a-R; CD88) on neutrophils and macrophages, leading to respiratory burst and release of bioactive cytokines and chemokines, which form the basis for its role during inflammation [2 ]. Using mouse strains that are deficient in C5 such as A/J and B10.osn (C5–/–), we showed previously that C5 deficiency affects the proinflammatory cytokine secretion of macrophages including TNF-{alpha}, IL-1ß, IL-6, IL-12, as well as chemokines (keratinocyte-derived chemokine, MIP-2, and MIP-1{alpha}) [3 ]. Macrophages were found to secrete a C5 peptidase, which cleaved C5 into C5a and C5b. C5a binds to the C5a-R in an autocrine manner to regulate several functions of macrophages. Addition of C5a peptide to C5–/– macrophages enhanced their bactericidal function and induced cytokine secretion, which was comparable with C5+/+ macrophages [3 ]. C5 deficiency led to defective granuloma formation, defects in macrophage-mediated killing of mycobacteria, and enhanced susceptibility to tuberculosis [3 , 4 ]. It is interesting to note that other C5-deficient strains such as DBA/2 and SWR have also been shown to be more susceptible to tuberculosis, although a causal link to C5 deficiency has not yet been shown for these strains [5 , 6 ]. Thus, we showed that in addition to its role in inflammation, C5a plays a major role in the control of macrophage-mediated control of infections.

C5a-R is a member of the rhodopsin family of seven-transmembrane G protein-coupled receptors [7 ]. C5a-R expression was earlier thought to be present only on the cells of myeloid origin such as granulocytes, monocytes/macrophages, and dendritic cells (DC) [8 9 10 ]. Recent studies have indicated the presence of C5a-R on nonmyeloid cells including epithelial cells and T and B lymphocytes [11 12 13 ]. However, there appears to be some discrepancy in the presence of C5a-R on murine T cells, and some studies indicate its presence using polyclonal antibodies to C5a-R, and some others fail to find it using an anti-C5a-R [14 ]. C5a-R expression on T cells is therefore an enigmatic issue. Recent studies show that during cutaneous hypersensitivity reactions in mouse ears, C5a is an early chemotactic peptide that recruits T cells to the site of inflammation [15 ]. The authors suggested that C5a is an early component of innate immunity, which is required for later elicitation of acquired T cell immunity [15 ]. As the role of C5a in macrophage-mediated control of tuberculosis has been described, its appears plausible that C5a may affect the function of T cells that are known to be essential for the regulation of macrophage function through cytokine and contact-dependent processes [16 ]. Because of the ambiguous findings on C5a-R expression of T cells, we have used novel models of activation to analyze receptor expression on murine T cells. We confirm in this study that naïve murine T cells lack measurable C5a-R but report a novel observation that APC induce an up-regulation of C5a-R in T cells. As macrophage or DC-mediated priming of T cells leads to acquired immunity, we suggest that the C5a peptide could play an additional role in the development of acquired immunity, modulating the T cell function through C5a-R.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacteria
Mycobacterium bovis bacillus Calmette-Guerin (BCG; #35734, American Type Culture Collection repository, Manassas, VA) was cultured in Middlebrook 7H9 broth to early log phase, harvested, and washed in sterile saline. Suspensions were then sonicated to disrupt large clumps and plated on 7H11 agar (Remel, Lenexa, KS) for CFU counts. Aliquots were then frozen at –70°C until one-time use.

Mice
Four- to 8-week-old, male or female B10.D2-H2dH2-T18cHc1/nSnJ (C5+/+), B10.D2-H2dH2-T18cHc0/oSnJ (C5–/–), or C57Bl/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were housed under specific pathogen-free conditions in accordance with Institutional Animal Care and Use Committee-approved procedures. For in vitro antigen-presentation assays, mice were immunized with BCG via i.p. injection, twice at weekly intervals, and spleens were used 1 week after the last dose. C5a-R-deficient (C5aR–/–) mice were generated by targeted deletion of the murine C5aR gene and determined to be completely C5aR–/– by RT-PCR, Northern blot, and immunohistochemistry analyses [17 ]. C5aR–/– mice, which had been back-crossed for over 10 generations with C57BL/6J mice, were used in all experiments.

Splenic T cells, macrophages, and DC
Spleens were minced and suspended in ACK lysis buffer for 5 min to lyse RBC, followed by two washes with PBS. A Pan T cell kit from Miltenyi Biotec (Auburn, CA) was used for negative isolation of CD3+ T cells. Mouse bone marrow (BM) cells from naïve mice were collected and processed in ACK lysis buffer and PBS. The cells were then cultured in IMDM containing 10% heat-inactivated FBS, penicillin, gentamicin, recombinant mouse IL-4, and recombinant mouse GM-CSF (10 ng/mL each, Cell Sciences Inc., Canton, MA) for 5–7 days to obtain the DC and medium with only GM-CSF to derive macrophages. Culture-grown DC were purified to 95–98% using CD11c (N418) magnetic beads (Miltenyi Biotec). Adherent macrophages cultured in medium with GM-CSF were tested by flow cytometry to be CD11c– and F4/80+, and DC were CD11c+ but F4/80-negative. Macrophages or DC were infected with BCG or activated with LPS as indicated.

In vitro stimulation
CD3+ T cells from C5+/+ and C5–/– mice were pulsed with Con A (100 ng/mL, Sigma Chemical Co., St. Louis, MO) or solid-phase anti-CD3 (10 µg/mL, Caltag Laboratories, San Francisco, CA) and incubated for 6–72 h at 37°C and 5% CO2. In addition, cells were stimulated with a mix of recombinant mouse IL-12 (5 ng/mL) and IL-18 (50 ng/mL, R&D Systems, Minneapolis, MN) for varying periods. At specific time intervals, cells were removed and stained for phenotype and C5a-R. Supernatants were tested by sandwich ELISA for IFN-{gamma} (R&D Systems).

Antigen-specific stimulation
C5+/+ and C5–/– mice were immunized with M. bovis BCG, and their spleens were used as the source of immune T cells. Naïve T cells were from nonimmunized mice. BM macrophages were plated in replicates at a density of 2 x 106 cells per well in six-well tissue-cultures dishes in IMDM. Macrophages were then infected with BCG (multiplicity of infection, 1:1) for 24 h with gentle mixing at 37°C and 5% CO2 to obtain a uniform infection. Microscopy revealed macrophages to contain at least 1 CFU per cell. Preliminary experiments determined that higher doses of organisms per macrophage (or DC) did not make a difference in their ability to prime the T cells. Monolayers were then washed free of nonphagocytosed bacteria with warm T cell medium (DMEM 10% FBS, penicillin, and gentamicin, 50 µM 2-ME) three times. CD3+ T cells purified from BCG-immunized or naïve mice were counted and added to the plate at a macrophage:T cell ratio of 1:20. Thus, each macrophage well received 40 x 106 T cells in T cell medium. After incubation at 37°C and 5% CO2 for indicated intervals, T cells were collected and stained for phenotype and C5a-R. In addition, the culture supernatant was collected for IFN-{gamma} assay using sandwich ELISA. Separate experiments were conducted to determine the viability of macrophages in T cell medium using trypan blue assay, and they were routinely >90% viable. It should be noted that the IMDM (for macrophages and DC) and DMEM used for T cells were comparable in composition. An identical experiment was conducted as a negative control using macrophages and T cells from C5a-R knockout (KO) mice.

DC-T cell cocultures
The method described for DC-T cell coculture, which results in Th1 expansion of naïve T cells, was followed [18 ]. DC were tested initially for C5a-R as naïve cells or after infection with BCG using anti-C5a-R as below. DC were then infected with BCG (1:1), washed after 4 h, and then plated in replicates at 2 x 106 cells/well using six-well plates. Purified CD3+ T cells from naïve C5+/+ and C5–/– mice were activated for 1 day with anti-CD3, rested for 1–2 days, and plated with DC in the presence of 10 ng/mL IL-2 per ml for 5 consecutive days at a DC:T cell ratio of 1:20. Thus, 40 x 106 T cells were added per well of DC in replicate cultures. T cells were harvested from cocultures, fractionated on Ficoll-Hypaque columns to remove dead cells at the end of 5 days, and tested for C5a-R as well as intracellular IFN-{gamma}. To control for antigen presentation for macrophages and DC cocultures, separate experiments were set up exactly as described, and the difference was that the macrophages and T cells were separated by a transwell culture insert. This prevents cell contact between macrophages and T cells but allows free flow of media and cytokines between the chambers. T cells were harvested and stained as above.

Flow cytometry
T cells, macrophages, or DC were suspended in PBS with 0.1% BSA and stained at 4°C using primary antibodies or isotype controls conjugated to fluorochrome. Mouse monoclonal anti-C5a-R has been characterized previously [14 ]. The intracellular staining was performed as follows: T cells harvested from cocultures were treated for 4 h with Golgi Stop (3 µM monensin, BD Biosciences, San Jose, CA) to block protein secretion from cells. T cells were added with FcR block (CD16/32, Caltag Laboratories) and incubated on ice for 15 min. The appropriate antibodies were added to each sample (CD3{epsilon}, Clone 145-2C1, Caltag Laboratories; CCD4, Clone L3T4, Caltag Laboratories; CD8{alpha}, Ly-2, Caltag Laboratories) and incubated for 30 min in the dark. The cells were washed and fixed with 2% paraformaldehyde for 30 min, followed by staining with anti-IFN-{gamma} FITC (LMR-9001-3, Caltag Laboratories), diluted in a perm buffer containing 0.1% Triton-X 100, 0.1% saponin, and PBS for 18 h at 4°C. The samples were washed in PBS, and cells were analyzed using a BD FACSort and CellQuest software (BD Biosciences).

Mouse infections with BCG
C5+/+ and C5–/– mice were infected i.p. with 106 CFU BCG each, and mice were killed at weekly intervals until Day 21. Spleens were harvested and immunophenotyped for CD3+ T cells expressing C5a-R as above. Infections and plating for CFU were done as per methods standardized for tuberculosis in mice [3 , 19 ].

Statistical analysis
All experiments were performed at least three times, and each experiment had triplicates of cultures per dose of activator. Significance was tested using Student’s t-test, and P values <0.05 were considered as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of C5a-R on naïve, Con A, or cytokine-activated T cells
Complete deficiency of C5 in mice is caused by a 2-bp gene deletion in a 5'-exon [20 ]. As C5a is produced on proteolytic cleavage of its parent molecule, C5, C5–/– mice lack the ability to generate C5a. To determine whether C5a-R expression was affected by the absence of C5, T cells from C5+/+ and C5–/– congenic strains of mice were analyzed in all experiments with an occasional use of T cells from C57BL/6 mice. Spleen-derived CD3+ T cells of mice were purified and tested for C5a-R as naïve or activated T cells. Naïve cells and those activated with Con A or PMA/ionomycin (data not shown) for 18 h did not show significant levels of C5a-R (Fig. 1a and 1b ). Cells were then activated with a mix of IL-12 and IL-18, and C5a-R expression continued to remain at low levels (Fig. 1c) . IL-12 and IL-18 have been used traditionally to induce IFN-{gamma} response in T cells [21 ]. Thus, to confirm that T cells were activated, IFN-{gamma} levels were measured by ELISA. Preliminary dose-response experiments were performed with 1, 5, 10, and 20 ng/mL each IL-12 and IL-18 to stimulate T cells. IFN-{gamma} levels matched the dose, and a dose of 10 ng/mL each was used as reported [21 ]. Figure 1d shows that Con A as well as IL-12 + IL-18 induced a strong release of IFN-{gamma}, suggesting that the lack of expression of C5a-R was not a result of the lack of stimulation of T cells.


Figure 1
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  		Figure 1. Expression of complement anaphylatoxin C5a-R on naïve or activated T cells derived from C5-deficient B10.oSnJ (C5–/–) and B.10.nSnJ (C5+/+) mice. Spleen-derived CD3+ T cells of mice were processed as below, stained for C5a-R-FITC, and immunophenotyped. T cells were tested as (a) naïve, (b) activated with Con A (100 ng/mL) for 18 h, or (c) activated with 5 ng/mL IL-12 and 50 ng/mL IL-18 for 18 h. Data represent three independent experiments. (d) Lack of C5a-R is not a result of lack of activation, as stimulation results in IFN-{gamma} secretion.

 
Expression of C5a-R on T cells following CD3 ligation or macrophage-mediated antigen presentation
Two kinds of experiments were performed. In the first, CD3+ T cells were activated with solid-phase anti-CD3 (Fig. 2a ), and anti-CD3 ligation resulted in an insignificant enhancement of C5a-R, which was not statistically significant (left panel), although copious IFN-{gamma} release was induced (right panel). In the next experiment, presensitized, splenic T cells from BCG-immune mice were cultured with BCG-infected macrophages. C5a-R was up-regulated by macrophage-induced priming. As early as 24 h postoverlay, there was an increase in C5a-R levels of C5+/+ and C5–/– T cells. By 72 h postpriming, C5a-R levels increased up to 15% of CD3+ T cells in both strains of mice (*, P<0.009; **, P<0.01, vs. 24 h, Fig. 2b , left panel). Macrophage-mediated priming was also accompanied by a strong release of IFN-{gamma} (Fig. 2b , right panel). Immune C5+/+ and C5–/– T cells overlaid on naïve macrophages did not show an up-regulation of C5a-R and produced <100 pg/mL IFN-{gamma} on their own (not shown). Control cultures were performed where macrophages were separated from T cells using transwells. The T cells from these physically separated cultures did not show appreciable levels of C5a-R (not shown). This suggests that culture supernatants from macrophages do not induce C5a-R. Finally, T cells from BCG-immunized mice were tested as negative controls, and they remained negative for C5a-R by flow cytometry (Fig. 2c) .


Figure 2
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  		Figure 2. CD3 activation and APC induce C5a-R in T cells. Splenic T cells were activated with solid-phase anti-CD3, or sensitized, splenic T cells from M. bovis BCG-immune mice were overlaid on BCG-infected macrophages as APC. (a) C5a-R is up-regulated moderately after CD3 ligation, which is also associated with an increase in IFN-{gamma}. (b) APC induce higher levels of C5a-R in immune T cells (*, P<0.009; **, P<0.01, vs. 24 h, left panel) accompanied by IFN-{gamma} secretion (right panel). Immune C5+/+ and C5–/– T cells overlaid on naïve macrophages do not show an up-regulation of C5a-R. T cells separated from macrophages in transwell cultures do not show C5a-R (not shown). (C) C5a-R knockout mice do not express C5a-R during co-culture.

 
DC induced up-regulation of C5a-R
When primed with mycobacterial antigen, DC prime naïve CD4 T cells to differentiate into IFN-{gamma}-secreting Th1 CD4 T cells in vitro [22 , 23 ]. As CD4 and CD8 can be displayed by some types of DC, we tested the CD3+ T cells alone for C5a-R expression in the following experiments: Initial studies examined the expression of C5a-R on DC alone. Figure 3a illustrates that DC express C5a-R when they are naïve (shaded) and show an up-regulation when infected with BCG. Figure 3b presents data from three independent experiments expressed as percent CD11c+ DC positive for C5a-R.


Figure 3
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  		Figure 3. DC express C5a-R. (a) BM-derived DC from C57Bl/6 (red), C5–/– (blue), and C5+/+ (green) strains of mice were tested with naïve (left) or after BCG infection (right) for C5a-R. DC express minimal C5a-R, but BCG infection up-regulates C5a-R in DC derived from all three strains of mice. (b) Data from three experiments shown (*, **, P<0.009, vs. naïve DC, t-test).

 
DC-induced priming of T cells induces C5a-R
BCG-infected DC were then cocultured with nonimmune, CD3-activated T cells for 5 days in the presence of IL-2. Figure 4a illustrates the expansion of CD3+ T cells induced to secrete IFN-{gamma} 24 h postpriming. Figure 4b shows that DC induced a copious secretion of IFN-{gamma} over time in the cocultures, which was a result of T cells, as DC alone secreted minimal IFN-{gamma}. C5–/– T cells produced less IFN-{gamma} than C5+/+ T cells in this coculture model. Although CD3 activation by itself induces IFN-{gamma} secretion in T cells (not shown), we used T cells that were activated with anti-CD3 but rested for 2 days in IL-2-containing medium for priming experiments with DC. Thus, when they were cultured alone (Fig. 4b) , they produced less IFN-{gamma}. Figure 5 illustrates that DC-induced expansion of T cells was associated with a marked increase in C5a-R levels. CD3+ T cells derived from cocultures were stained for C5a-R, and as much as 50% of T cells expressed C5a-R. Physical separation of DC and T cells results in lack of C5a-R expression in T cells (not shown).


Figure 4
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  		Figure 4. BCG-primed DC induce differentiation of CD3+ IFN-{gamma}+ cells. (a) DC from C5–/– and C5+/+ mice were cocultured with anti-CD3-activated, nonimmune T cells from C5–/– and C5+/+ mice. IFN-{gamma} levels were measured over time. T cells cocultured with DC were stained for intracellular IFN-{gamma}, and a typical profile of CD3+ IFN-{gamma}+ cells representing three experiments is shown. (b) Production of IFN-{gamma} is shown from triplicate experiments. DC prime T cells from both strains of mice to secrete IFN-{gamma}, although C5–/– T cells generally produce less IFN-{gamma} than C5+/+ T cells.

 

Figure 5
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  		Figure 5. BCG-primed DC induce an up-regulation of C5a-R on murine T cells. IFN-{gamma}+ T cells from Figure 4 were tested for C5a-R. BCG-infected DC prime a large increase in the expression of C5a-R in T cells. Cytometric profile is illustrated to the left, and data from triplicate experiments are shown to the right (*, **, P<0.007, vs. naïve T cells). T cells cocultured with naïve DC do not show an up-regulation of C5a-R.

 
C5a-R in T cells in vivo
Mice were infected with BCG and tested at weekly intervals for C5a-R expression on splenic T cells. C5–/– and C5+/+ spleens contained ~25% CD4+ and CD8+ T cells each per spleen over 3 weeks of infection, which did not increase. CD3+ T cells of the same spleens showed progressively increasing numbers of C5a-R+ T cells (Fig. 6 ). Naïve, spleen-derived T cells did not show C5a-R. Colony counts of BCG showed that the bacteria increased slowly in the spleens over 3 weeks, from 103 per spleen to 104 per spleen by 3 weeks (data not shown). These results suggest that the increased expression of C5a-R was most probably a result of increased local priming over time.


Figure 6
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  		Figure 6. BCG infection of mice induces an up-regulation of C5a-R on splenic T cells. Mice were infected i.p. with BCG at 106 CFU per mouse. Splenic T cells were typed for CD4+ and CD8+ T cells and for CD3+ T cells expressing C5a-R over 3 weeks after i.v. infection. C5a-R+ T cells increase in numbers over 3 weeks (a). Naïve mice do not show appreciable levels of C5a-R+ CD3+ T cells. Cytometric expansion of C5a-R+ CD3+ T cells is illustrated (b). C5–/– and C5+/+ spleens have comparable numbers of CD4+ and CD8+ T cells over 3 weeks, and a Day 21 profile is illustrated (b, upper left). Cytometry of C5a-R+ CD3+ T cells in spleens of C5+/+ mice over 3 weeks of infection is illustrated in other panels.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although C5a-R expression on myeloid cells has been well characterized, its distribution on T cells appears debated [14 , 24 , 25 ]. A polyclonal antibody was used to identify C5a-R on human T cells [12 ]. However, using mAb or using FITC-labeled C5a peptide, it has been difficult to detect C5a-R on T cells [14 ]. Using the 20/70 mAb specific to C5a-R, such as the one used in this study, C5a-R was found to be expressed on murine myeloid cells but not on naïve T cells from spleens or those activated in vitro [14 ]. We used congenic mice sufficient and deficient for C5 to study the distribution of C5a-R, and in general, both mice showed similar expression of C5a-R. Limited experiments were carried with C57Bl/6 mice, which suggested that they were similar to these B10 mice (not shown). This study reproduced the findings of Soruri et al. [14 ], in that naïve T cells or those activated with Con A were negative for C5a-R. It is interesting that T cells stimulated with IL-12 and IL-18 produced copious IFN-{gamma} but were still negative for C5a-R (Fig. 1c and 1d) . That cytokine-treated T cells did not express C5a-R was unanticipated, as when macrophages and DC prime presensitized T cells or naïve T cells, they secrete IL-12 and IL-18 [26 , 27 ]. These cytokines in turn induce IFN-{gamma} from T cells through the STAT-4 signaling pathway and trigger development of Th1 responses and/or induce proliferation of T cells [21 ]. Previous reports indicate that PHA failed to induce C5a-R, and our study confirms that activation with Con A, PMA, IL-12, or IL-18 also failed to do so. Together, these studies indicate that purified T cells isolated from spleens or those activated in ex vivo culture do not show adequate expression of C5a-R. Thus, expression of C5a-R on T cells appears to be unrelated to activation induced by conventional methods.

Some studies in the past have shown that C5a-R can be detected in situ in mouse or human tissues [28 29 30 ]. This led us to hypothesize that optimal C5a-R expression on T cells may depend on conditions that may occur in vivo. When T cells are primed by macrophages or DC in vivo, they cross-talk with T cells through a TCR-MHC molecular complex, which consists of antigen-presenting (MHC II, CD1d), costimulatory (CD80, CD86), and various adhesion molecules (CD40, CD44, CD11a). We theorized that such a cross-linking mechanism is probably necessary to optimally prime and activate T cells. Although the interaction among macrophages, DC, and T cells is governed by a complex environment in vivo, it is possible to duplicate such experiments in vitro to a certain extent. Thus, macrophages have been used extensively to prime presensitized T cells in vitro, and DC cocultured with naive T cells have been shown to drive them to Th1, Th2, or Th3 cytokine-producing T cells in ex vivo models [18 , 22 ]. We sought to evaluate the effect of macrophages and DC on C5a-R expression using similar models.

Initial studies showed that activation of T cells with anti-CD3 alone induced insignificant levels of C5a-R compared with unprimed cells (Fig. 2a) . This indicated that CD3 ligation alone is not enough for induction of C5a-R on T cells. Next, macrophages were allowed to prime presensitized T cells, which produced significant IFN-{gamma} following priming, and there was a significant increase in the level of C5a-R. Figure 2b shows that as early as 24 h postpriming, nearly 5% of T cells had C5a-R, and this level increased up to 15% by 72 h postoverlay. C5–/– as well as C5+/+ mice showed comparable levels of C5a-R. As CD3 ligation alone failed to induce C5a-R, but cross-bridging with macrophages induced C5a-R, we suggest that the macrophage-T cell contact through a MHC-TCR complex is necessary for the induction of C5a-R in T cells. Furthermore, physical separation of T cells and macrophages did not induce appreciable levels of C5a-R. Finally, T cells obtained from C5a-R KO mice were negative for C5a-R despite APC-induced priming, suggesting the staining with the mAb for C5a-R was specific. Additional evidence was generated using DC.

During the initiation of a primary immune response, DC prime naïve T cells in the lymph nodes and cause them to develop into effector T cells capable of recognizing antigen presented through the MHC II complex on APC [22 ]. Thus, additional experiments focused on determining whether T cells express C5a-R after DC-induced priming. Initial studies mapped the expression of C5a-R on naïve or BCG-infected DC, which when derived from three strains of mice, expressed significant levels of C5a-R up-regulated after BCG infection. As BCG is known to induce maturation of DC, this suggested that C5a-R expression in DC is associated with the maturation process [31 , 32 ]. This finding is consistent with a previous report that maturation of DC is associated with expression of C5a-R and that splenic, immature DC did not show appreciable levels of C5a-R [14 , 30 , 33 ]. As our DC were derived from BM through GM-CSF and IL-4-induced differentiation, it can be argued that such cells are more activated than in situ-derived DC. However, previous studies have shown that BM-derived DC have a phenotype comparable with in vivo-derived DC, and our own preliminary studies have indicated that splenic DC as well as lung-derived DC isolated from mice show activation markers similar to those derived from BM when primed with BCG (supplemental data).

Coincident with the increased levels of C5a-R, DC infected with BCG induced expansion of CD3+ T cells that secreted IFN-{gamma} (Fig. 4a) . It is interesting that when such cells were tested for C5a-R, a large percentage of T cells was found to express C5a-R (Fig. 5) . Finally, mice were infected with BCG and C5a-R expression analyzed in vivo. There was a progressive increase in the numbers of C5a-R+ CD3+ T cells in the spleens of mice over 3 weeks. Although the reasons for the low number of C5a-R+ T cells early during infection are not clear, it must be noted that these mice received one injection of 106 CFU BCG. As BCG becomes distributed in various internal organs, the number of bacteria available to prime T cells in the spleen may have been proportionately low. Increase in numbers of BCG, which occurred over 3 weeks, may have led to an increase in the number of C5a-R+ T cells.

Although the results from this study are interesting, they also raise additional questions. DC were better in inducing C5a-R than macrophages in T cells in ex vivo models, which suggests that DC are probably better in priming T cells compared with macrophages. Conversely, there may be a need for naïve T cells to express C5a-R early during sensitization. The early recruitment effect of C5a during cutaneous hypersensitivity supports this concept [15 ]. The low levels of C5a-R in mice compared with in vitro-primed T cell levels are also intriguing. It is possible that higher levels of C5a-R are expressed early after infection so that limitations in screening procedures may not adequately detect in vivo C5a-R expression. Finally, C5a-R+ T cells may belong to several effector classes that need to be characterized to understand the significance of C5a-R expression.

In summary, using in vitro-priming systems, one based on macrophages and another using DC, we have shown that antigen-specific T cell activation results in an up-regulation of C5a-R on CD3+ T cells. The presence of C5a-R+ on T cells has also been confirmed during infection in vivo. These novel observations suggest a new role for C5a–C5a-R interactions in the development of an immune response. We propose that up-regulation of C5a-R during T cell priming may enable the T cells to receive additional signals through soluble C5a peptide released from APC, and this may affect the development of T cells into different types of effectors.


   ACKNOWLEDGEMENTS
 
The authors thank the National Heart, Lung, Blood Institute, National Institutes of Health (Bethesda, MD), for providing support for this study through Grant HL68520 to C. J. and HL074333 to R. A. W. and to the National Institute of Allergy and Infectious Diseases through Grant AI025011 to R. A. W. We are grateful to Dr. Jorg Zwirner (George August University, Gottingen) for the mAb against C5a-R.

Received October 15, 2005; revised June 25, 2006; accepted July 20, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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