Interaction of Macrophage Antigen 1 and CD40 Ligand Leads to IL-12 Production and Resistance in CD40-Deficient Mice Infected with Leishmania major

Numerous studies have shown that CD40–CD40L interaction is essential for induction of effective cell-mediated immunity to pathogens. CD40 is constitutively expressed on basophils, dendritic cells (DCs), B cells, and epithelial cells but can be induced on macrophages, endothelial cells, smooth muscle cells, and fibroblasts. CD40L, in contrast, is inducible on CD4⁺ and CD8⁺ T cells, B cells, epithelial cells, eosinophils, monocytes, macrophages, NK cells, and mast cells (1). Previous reports show that CD40–CD40L interaction is critical for the production of IL-12 and induction of optimal Th1 response and immunity to cutaneous leishmaniasis (2). Treatment of the susceptible BALB/c mice with CD40 agonistic Ab led to healing postinfection with Leishmania major (3). In contrast, injection of anti-CD40L antagonists led to susceptibility in the resistant C57BL/6, which was associated with reduced IL-12 production (4). Furthermore, deficiency of CD40L or CD40 in the usually resistant C57BL/6 leads to susceptibility to L. major (5, 6) and Leishmania amazonensis (7) infections. Interestingly, some studies suggest that CD40–CD40L interaction is not required for protection against L. major (8, 9). In addition, blockade of CD40–CD40L interaction in patients with cutaneous leishmaniasis due to Leishmania braziliensis did not affect IFN-γ production by T cells or progression of cutaneous lesion. Thus, the role of CD40–CD40L interaction in leishmaniasis remains controversial and needs to be further delineated.

Macrophage Ag 1 (Mac-1) is a β2 integrin that is present on monocytes, neutrophils, and macrophages (10), and it plays an essential role in immunity by influencing adhesion and migration of phagocytic cells (11). It is composed of two chains, CD18 and CD11b, a type 1 transmembrane receptor composed of extracellular, transmembrane, and cytoplasmic domains (12). The cytoplasmic domain of CD11b is believed to be important for recognition of pathogen-associated molecular patterns such as Mycobacterium tuberculosis oligosaccharides and Leishmania lipophosphoglycan (13). In contrast, the I domain near the N-terminal region is responsible for binding C3bi, fibrinogen, and other bacterial Ag (14). The binding ability of the C and I domains could explain the wide range of ligands bound by Mac-1 (14, 15). Recent reports show that, in the absence of CD40, Mac-1 can bind to CD40L and mediate the production of proinflammatory cytokine leading to inflammation (10). However, whether Mac-1 can bind to CD40L and regulate the outcome of immunity to L. major has not yet been determined.

In this article, we compared the immune response and outcome of L. major infection in CD40 and CD40L knockout (KO) mice and investigated the role of Mac-1–CD40L interaction in protective immune response to L. major. Our data reveal striking differences in disease progression and immune response in IL-12–treated CD40 and CD40L KO mice infected with L. major. Whereas rIL-12–treated CD40 KO mice remained healed, developed long-term immunity, and were resistant to secondary L. major challenge, rIL-12–treated CD40L KO reactivated their lesion after cessation of treatment. We show that these differences in disease pathogenesis were related, in part, to alternative utilization of CD40L–Mac-1 pathway for continuous and sustained IL-12 production in CD40 KO mice. Thus, our studies reveal a critical but redundant role of Mac-1–CD40L interaction in IL-12 production and maintenance of optimal immunity to L. major infection.

Six- to eight-week-old female wild type (WT) C57BL/6 mice were purchased from Charles River Laboratories (St-Constante, QC, Canada). CD40- and CD40L-deficient mice on C57BL/6 background were purchased from The Jackson Laboratory (Sacramento, CA). Mice were housed in specific pathogen-free units at the Central Animal Care Services, University of Manitoba. All mouse experiments were approved by the University of Manitoba Animal Care Committee in accordance with the regulation of the Canadian Council on Animal Care.

L. major parasites (MHOM/IL/80/Friedlin) were grown in Grace’s insect medium (Life Technologies) supplemented with 20% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. For infection, 7-d stationary-phase promastigotes were washed three times in PBS and counted. Mice were infected by injecting 10⁶ parasites suspended in 50 μl PBS into the right hind footpad. Lesion development and progression were monitored weekly by measuring the diameter of the infected and uninfected footpads with Vernier calipers.

To obtain healed WT, we monitored infected mice (as described earlier) until their cutaneous lesions were completely resolved (>12 wk). To generate healed CD40 and CD40L KO mice, we infected CD40 and CD40L KO mice as described earlier and treated with rIL-12 (0.3 μg) intralesionally three times per week for the first 2 wk of infection. This treatment leads initially to complete resolution of lesions in both KO mice, which is sustained only in CD40 KO mice, whereas disease recrudescence occurs in CD40L KO mice from 7 wk postinfection (see 16Results). In some experiments, rIL-12 treatment was continued until week 7 or restarted at weeks 5–7 postinfection (after the first 0–2 wk treatment) in the CD40L KO mice.

Healed WT and CD40 KO mice were challenged with 5 million L. major in the contralateral footpad (left foot). Challenged mice were assessed for delayed-type hypersensitivity (DTH) response at 72 h postchallenge by measuring the thickness of the challenged footpads with Vernier calipers. Unchallenged footpads were used as controls. Challenged mice were sacrificed at 3 wk postchallenge, and parasite burden was determined by limiting dilution analysis as previously described (16).

To determine whether Mac-1 signaling is important for the maintenance of immunity in healed WT or CD40 KO mice, we injected anti–Mac-1 (clone M1/70) Ab or control Ig (human IgG1 100 μg/mouse) i.p. at 8 wk postinfection, and lesion recrudescence was monitored weekly. To investigate the role of Mac-1 signaling in secondary (memory) anti-Leishmania immunity, we injected healed (>12 postinfection) WT and CD40 KO mice with anti–Mac-1 mAb (100 μg/mouse) and then challenged them with L. major in the contralateral footpad the next day. Challenged mice were sacrificed after 3 wk to determine parasite burden and immune response.

At various times postinfection, infected mice were sacrificed and the draining popliteal lymph nodes were harvested and made into single-cell suspensions. Cells were washed, suspended at 4 million/ml in complete medium (DMEM supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin), and plated at 1 ml/well in 24-well tissue culture plat (Falcon, VWR Edmonton, AB, Canada). Cells were stimulated with soluble Leishmania Ag (SLA; 50 μg/ml) for 72 h, and the supernatant fluids were collected and stored at −20°C until assayed for cytokines by ELISA.

IL-12p40, IFN-γ, and IL-4 concentrations in cell culture supernatant fluids were measured by sandwich ELISA using Ab pairs from BD Pharmingen (San Jose, CA) according to manufacturer’s suggested protocols. The hybridoma clones for the Ab pairs are: IL-12p40, C15.6 and C17.8; IFN-γ, R4-6A2 and XMG1.2; and IL-4, 11B11 and BVD6-24G2.

Bone marrow–derived DCs (BMDCs) and bone marrow–derived macrophages (BMDMs) were generated from naive WT, CD40, and CD40L KO mice as described previously (17). In brief, bone marrow cells were isolated from the femur and tibia of mice and differentiated into macrophages using complete medium supplemented with 30% L929 cell culture supernatant. BMDCs were differentiated in petri dishes in the presence of 20 ng/ml rGM-CSF (Peprotech, Rocky Hill, NJ). Immature DCs were harvested on day 7 and assessed for the expression of CD11c, CD40, CD80, CD86, and MHC class II by flow cytometry.

Splenic CD90.2⁺, CD11b⁺, and CD11c⁺ cells were isolated by positive selection using StemCell CD90.2⁺, CD11b⁺, and CD11c⁺ cells EasySep isolation kits, respectively, according to the manufacturer’s suggested protocols. The purities of the different cell populations were CD90.2⁺ (98%), CD11b⁺ (94%), and CD11c⁺ (87–93%).

Splenic CD90.2⁺ T cells were activated in vitro by stimulating with soluble anti-CD3ε and anti-CD28 (2 μg/ml) mAbs overnight. The next morning, the cells were washed and then cocultured with splenic CD11c⁺ or CD11b⁺ cells at 100 T cells to 1 CD11c⁺ or CD11b⁺ cells (100:1) for 48 h.

BMDMs, BMDCs, and splenic CD11b⁺ and CD11c⁺ cells (2 × 10⁶ cells/ml were cultured in 96-well flat-bottom tissue culture plates (100 μl/well; Falcon) in the presence or absence of anti–Mac-1 Ab (5 μg/ml). In some experiments, some wells were stimulated with sCD40L (2 μg/ml), anti-CD40 mAb (5 μg/ml), or LPS (1 μg/ml). After 48 h, the culture supernatant fluids were collected and assayed for IL-12 by ELISA.

Healed WT and CD40 KO mice were injected with anti–Mac-1 mAb or control Ig (100 μg/mouse) and then challenged with 5 million L. major in the contralateral footpad the next day. After 7 d, mice were sacrificed and mononuclear cells were isolated from the footpads as previously described (18). The cells were stained directly ex vivo with fluorochrome-conjugated Abs against CD3, CD4, CD8, and CD11b, and the expression of these markers was assessed by flow cytometry.

Student t test was used to compare the mean and SEM between two groups. In some experiments, nonparametric one-way ANOVA was used to compare mean and SD of more than two groups. Tukey’s test was used where there was a significant difference in ANOVA. Differences were considered significant when p ≤ 0.05.

IL-12 produced by DCs is important for the development of naive CD4⁺ T cells into Th1 cells that mediate and maintain immunity in cutaneous leishmaniasis (19–21). Although some studies show that CD40–CD40L interaction is critical for IL-12 and the development of protective anti-Leishmania immunity (7), others show that CD40L-deficient mice are capable of producing IL-12 and mounting a protective Th1 response against L. major infection (8, 9). To fully determine the role of CD40 and CD40L in IL-12 production, and thus resistance to experimental L. major infection, we compared disease progression and immune response in CD40 and CD40L KO infected with L. major. Consistent with previous reports, both CD40 and CD40L KO mice were highly susceptible to infection as evidenced by their inability to control lesion development (Fig. 1A, 1B) and parasite replication (Fig. 1C). Treatment of infected CD40 and CD40L KO mice with rIL-12 during the first 2 wk of infection led to healing (lesion resolution and parasite control) in both CD40 and CD40L KO mice (Fig. 1A, 1B). However, whereas treated CD40 KO mice remained resistant for >14 wk, treated CD40L KO mice reactivated disease starting around week 5–7 postinfection (Fig. 1A, 1B). The impaired parasite control and lesion recrudescence in rIL-12–treated CD40L KO mice corresponded with significantly (p < 0.01–0.001) lower IL-12 (Fig. 1D) and IFN-γ (Fig. 1E) and significantly (p < 0.001) higher IL-4 (Fig. 1F) production by their draining lymph node cells compared with the CD40 KO or WT mice. Interestingly, although continuous treatment of infected CD40L KO mice with rIL-12 resulted in lesion control and prevented disease reactivation, restarting rIL-12 treatment at weeks 5–7 after 3–4 wk cessation failed to prevent disease reactivation (Fig. 1G). Because continuous IL-12 is required for maintenance of anti-Leishmania immunity in healed mice (19–21), these observations suggest the existence of an alternative pathway for IL-12 production in CD40 KO mice that is nonfunctional or absent in CD40L KO mice. They further suggest that in the infected CD40L KO mice, exogenous IL-12 treatment is important for initiating a protective immune response but is unable to prevent disease progression once lesion has developed.

DCs and macrophages are the major cell types that produce IL-12 during Leishmania infection (2, 22). Previous reports show that Mac-1 binds to CD40L and this interaction mediates inflammation (10) and leukocyte recruitment to atherogenic sites (23). Because we observed that early treatment with rIL-12 results in long-term healing in CD40 KO mice, but not CD40L KO mice, we hypothesized that the sustained resistance observed in CD40 KO mice is due to IL-12 production via the interaction of Mac-1 with CD40L. Therefore, we purified CD11b⁺ and CD11c⁺ cells from WT, CD40, and CD40L KO mice and stimulated them with soluble CD40L. As expected, stimulation of cells from WT and CD40L KO mice (which express intact CD40 molecules) led to IL-12 production (Fig. 2A, 2C, 2D, 2F). Interestingly, sCD40L stimulation also led to IL-12 production (Fig. 2B, 2E) in CD40 KO cells, suggesting that sCD40L must be binding to another molecule that is distinct from CD40. The ability of sCD40L to induce IL-12 production in cells from CD40 KO mice was significantly (p < 0.05–0.001) blocked by the addition of anti–Mac-1 blocking Ab (Fig. 2B, 2E). Similar results were also obtained with BMDMs and BMDCs (Supplemental Fig. 1). Interestingly, anti–Mac-1 mAb has no effect on LPS-induced IL-12 production by CD11c⁺ and CD11b⁺ cells from CD40 KO mice (Fig. 2B, 2E), suggesting that this effect is specific to sCD40L stimulation. Taken together, these findings suggest that, in the absence of CD40, Mac-1 can interact with CD40L leading to IL-12 production, and this may account for the difference in the outcome of infection after rIL-12 treatment in the CD40 KO and CD40L KO mice infected with L. major.

The preceding results show that anti–Mac-1 blocking Ab inhibits sCD40L-induced IL-12 production in vitro. Although CD40L is mainly expressed by activated CD4⁺ T cells, other cell types including eosinophils, basophils, mast cells, DCs, macrophages, and NK cells are also known to express CD40L under certain conditions (24). Previous reports show that the production of IL-12 by DCs during Leishmania infection is dependent on the interaction of CD40 molecules (on DCs) with CD40L expressed on activated CD4⁺ T cells (2, 25, 26). Therefore, we assessed whether anti–Mac-1 blocking Ab could inhibit activated CD4⁺ T cell–induced IL-12 production by DCs in vitro. As for sCD40L, activated CD4⁺ T cells induced robust IL-12p40 production by BMDCs from WT, CD40, or CD40L KO mice in vitro (Fig. 3A–C). Interestingly, although anti–Mac-1 mAb inhibited CD4⁺ T cell–induced IL-12p40 production by DCs from CD40 KO mice (Fig. 3B), it has no effect on IL-12p40 production by DCs from WT and CD40L KO (Fig. 3A, 3C), suggesting that binding of CD40L on T cells to Mac-1 on DCs is responsible for IL-12p40 production in CD40 KO cells. To further confirm that the interaction of T cell–expressed CD40L with Mac-1 on DCs leads to IL-12p40 production, we cocultured DCs from WT mice with activated T cells from WT, CD40 KO, and CD40L KO mice and assessed IL-12p40 production by ELISA. Unlike activated T cells from WT and CD40 KO mice that induced robust IL-12p40 production, CD40L KO T cells were unable to induce IL-12p40 production in DCs in vitro (Fig. 3D), akin to anti–Mac-1 blockade. Collectively, these results suggest that CD40L–Mac-1–induced IL-12 production is a redundant pathway that is operational only in the absence of functional CD40 molecule. Thus, in the presence of intact CD40 signaling as in WT mice, CD40L–Mac-1 interaction is dispensable for IL-12p40 production by DCs. Interestingly, the expression of CD40L on T cells from L. major–infected CD40 KO mice was upregulated after rIL-12 treatment (Supplemental Fig. 2). This observation suggests that exogenous IL-12 is required for initiating the CD40L–Mac-1 redundant pathway of resistance in L. major–infected CD40 KO mice.

The preceding observations show that IL-12 production in a Mac-1–dependent manner is responsible for maintaining resistance in CD40 KO mice. We reasoned that if resistance in IL-12–treated CD40 KO mice is dependent on IL-12 production through Mac-1 pathway, blockade of Mac-1 in these mice should lead to impaired IL-12 production and disease reactivation. Therefore, we injected anti–Mac-1 blocking mAb or control Ig into healed CD40 KO and WT mice and monitored the treated mice over time for disease reactivation. As shown in Fig. 4A, CD40 KO treated with anti–Mac-1 mAb spontaneously reactivated their cutaneous lesion within 1 wk posttreatment. This was associated with significantly (p < 0.01) higher parasite burden compared with those that received control Ig (Fig. 4B). Disease reactivation in anti–Mac-1–treated healed CD40 KO was also associated with significantly (p < 0.01 and p < 0.001) lower IL-12 (Fig. 4C) and IFN-γ (Fig. 4D) production by cells from the draining lymph nodes compared with those that received control Ig. In contrast, cells from the draining lymph nodes of anti–Mac-1–treated CD40 KO mice produced significantly higher levels of IL-4 than those given control Ig (Fig. 4E). In contrast, anti–Mac-1 mAb treatment did not result in disease reactivation, increased parasite burden, or altered cytokine production in healed WT mice (Fig. 4). Collectively, these results support our conclusion that Mac-1–dependent IL-12 production is responsible for the maintenance of resistance in IL-12–treated CD40 KO mice.

As an integrin, Mac-1 plays an important role in adhesion and migration of cells into infection sites (11). Therefore, we wished to determine whether disease reactivation in healed CD40 KO mice after anti–Mac-1 Ab treatment was due to alteration in cell composition and/or migration into the infection site in healed CD40 KO mice. Healed WT and CD40 KO mice were injected with Mac-1 blocking Ab, challenged with L. major after 24 h, and sacrificed 6 d later to assess cellular composition in the footpads. There was no significant difference in the percentages of CD11b⁺ (Fig. 5A, 5B) and CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells (Fig. 5C–E) in the footpads of both healed CD40 KO and WT mice treated with anti–Mac-1 or control Ig. Taken together, these results suggest that disease reactivation in healed CD40 KO after blockade of Mac-1 is not related to alteration in cell composition and/or migration into the infection sites.

Next, we investigated the impact of Mac-1 blockade on secondary immunity to L. major. Healed (>14 wk postinfection) CD40 KO and WT mice were treated with anti–Mac-1 blocking Ab 24 h before challenge with virulent L. major. There was no significant difference in DTH response and rapid control of parasite in healed WT mice treated with either anti–Mac-1 mAb or control Ig (Fig. 6A). In contrast, although healed CD40 KO mice treated with control Ig displayed strong DTH response and rapid parasite control akin to WT mice, CD40 KO mice treated with anti–Mac-1 mAb blocking Ab had significantly (p < 0.001) lower DTH response (Fig. 6A), as well as significantly (p < 0.01) higher parasite burden (Fig. 6B). Further, the levels of IL-12 (Fig. 6C) and IFN-γ (Fig. 6D) in the supernatant fluid of cells from CD40 KO treated with anti–Mac-1 blocking Ab were significantly (p < 0.01) lower than those from the control Ig–treated group. Taken together, these results indicate that Mac-1 plays an important role in secondary immune response to L. major.

There have been conflicting reports on the role of CD40–CD40L interactions in immunity to experimental cutaneous leishmaniasis. Even though a study showed that CD40L KO mice heal their low-dose L. major infection (9), another study showed that these mice are highly susceptible to L. amazonensis infection (7). Therefore, the primary aim of this study was to more precisely determine the role of CD40 and CD40L interactions in primary and secondary immunity to L. major. We reasoned that if the interaction between CD40 and CD40L is the sole mechanism responsible for IL-12 production in L. major–infected mice, then the outcome of infection after rIL-12 treatment should be the same in infected CD40 and CD40L KO mice. Surprisingly, we found that there are differences in disease outcome and immune response between CD40 and CD40L KO mice infected with L. major after treatment with rIL-12. Whereas CD40 KO mice treated with rIL-12 during the first 2 wk of infection completely heal their footpad lesion and are resistant to secondary challenge, CD40L KO mice reactivated disease a few weeks after cessation of rIL-12 treatment. This inability to control infection in CD40L KO mice was associated with impaired IL-12 and IFN-γ production by cells from the spleens and lymph nodes draining the primary infection sites. Because continuous IL-12 production is indispensable for the initiation and maintenance of optimal cell-mediated immunity to L. major, the sustained immunity in rIL-12–treated CD40 KO mice suggests the existence of an alternative pathway for IL-12 production in these mice, which is absent or nonfunctional in CD40L KO mice. We showed that blockade of Mac-1 signaling using blocking anti–Mac-1 mAb in healed CD40 KO led to impaired IL-12 production, disease reactivation, and impaired Th1 response, suggesting that signaling via Mac-1 is responsible for IL-12 production and sustained immunity in CD40 KO mice. Indeed, we showed that anti–Mac-1 mAb blocked sCD40L-induced IL-12 production by macrophages and DCs from CD40 KO, suggesting that the interaction of Mac-1 with CD40 is responsible for IL-12 production in CD40 KO mice.

IL-12 is a critical cytokine for both the development of primary immunity and the maintenance of effector Th1 cells that mediate secondary resistance to L. major. Although it is widely believed that CD40–CD40L interaction is the primary pathway responsible for IL-12 production for optimal anti-Leishmania immunity, we and others have identified other alternative pathways such as TRANCE-RANK (27) and LIGHT-HVEM (28, 29) that contribute to IL-12 production and development of immunity to L. major. This raises the question as to which of the different pathways is the most critical during primary and secondary/memory phases of immunity to L. major. We believe that although all these other pathways may be important for optimal immunity to L. major, the current observations support the idea that in the absence of CD40–CD40L interaction, Mac-1–CD40L interaction plays an important role in this resistance. This is supported by the fact that neither blockade of TRANCE–RANK interaction nor inhibition of LIGHT signaling leads to spontaneous disease reactivation or loss of immunity in healed CD40 KO mice (data not shown). In contrast, blockade of Mac-1 led to both spontaneous disease reactivation (akin to IL-12 KO mice) and loss of infection-induced immunity in CD40 KO that healed their primary L. major infection.

Although our in vitro and in vivo studies strongly suggest that Mac-1 interaction with CD40L is responsible for sustained IL-12 production in mice lacking functional CD40, it is plausible that other as yet untested pathways may be involved. For example, it has been shown that sCD40L can bind to another integrin, α5β1 (30), which is a primary receptor for fibronectin, a key molecule that plays an important role in regulating inflammatory cytokine production by some cells (31, 32). However, we believe that Mac-1–CD40L interaction is responsible for the bulk of IL-12 produced in this system because addition of anti–Mac-1 mAb completely abolished IL-12 secretion by DCs and macrophages from CD40 KO mice after stimulation with sCD40L (Fig. 3). In line with this, activated T cells from CD40L KO mice were unable to induce IL-12p40 production in DCs from WT mice in vitro. Interestingly, rIL-12 treatment dramatically upregulated the expression of CD40L on CD3⁺ T cells from L. major–infected CD40 KO mice (Supplemental Fig. 2), suggesting that exogenous IL-12 may be required for initiating the CD40L–Mac-1 redundant pathway of resistance in L. major–infected CD40 KO mice.

Our findings that Mac-1–CD40L interaction enhances IL-12p40 production is in disagreement with recently published data showing that the uptake of Leishmania via Mac-1 leads to inhibition of IL-12 production in macrophages (33). This difference may be related, in part, to the experimental models and approach between these studies. In this study, DCs and macrophages were not infected with L. major in vitro. In addition, Ricardo-Carter et al. (33) merely assessed the effect of Mac-1– on LPS-induced IL-12 production in L. major–infected WT or Mac-1–deficient macrophages and did not assess the impact of Mac-1 deficiency on T cell induction of IL-12 production in infected cells. The Mac-1–dependent inhibition of LPS-induced IL-12p40 production in infected macrophages was mediated by intracellular signals involving the E26 transformation–specific pathway. It is plausible that Mac-1–CD40L interaction does not activate the E26 transformation–specific pathway, and thus would not inhibit IL-12 production. Although not assessed in this study, this proposal is supported by the recent report that shows the interaction of CD40L with all its receptors leads to activation of the MAPK pathway (34), which is a key intracellular signaling pathway that leads to IL-12 production in macrophages and DCs (35, 36).

Although Li and colleagues (37) reported that stimulation of neutrophils with soluble CD40L led to their increased expression of Mac-1, we have not observed any difference in the level of Mac-1 (CD11b) expression on cells stimulated with sCD40L. This could be because the cells (macrophages) already expressed very high levels of CD11b (data not shown), and thus it was difficult to further increase their expression. de Oliveira and colleagues (38) recently showed that serum levels of sCD40L increase in VL patients who are responding positively to treatment, and they concluded that sCD40L could be used as a biomarker for monitoring treatment outcome in visceral leishmaniasis. Whether the serum level of sCD40L increases after experimental L. major infection in both WT and CD40 KO mice is unknown. Given that the interaction of sCD40L and Mac-1 is critical for maintaining anti-Leishmania immunity in CD40 KO mice, it is conceivable that the level of sCD40L in the serum or expression of CD40L on T cells is high in healed CD40 KO mice. However, more studies are needed to address this question.

Mac-1, also known as complement receptor 3, is a heterodimeric protein that consists of CD11 and CD18 molecules and plays multiple roles including immunity, adhesion, and cell migration. Mac-1 binds to an array of ligands including extracellular matrix proteins (39), ICAM-1 (40), bacterial LPS (41), and complement opsonized Leishmania lipophosphoglycans (42). Interestingly, the interaction of Leishmania to Mac-1 results in silent entry of the parasite into the phagocytes, thereby avoiding the activation and release of reactive oxygen intermediates and subsequent lysis of the parasites (43, 44). Also, a report showed that Mac-1–deficient C57BL/6 mice display similar lesions and parasite burden as WT mice postinfection with L. major (45). Although this observation is inconsistent with our findings, it is conceivable that Mac-1 is a functionally redundant pathway that becomes important only in the absence of CD40–CD40L interaction. Thus, during L. major infection, CD40-CD40L signaling is the primary pathway for IL-12 production. IL-12 production via the Mac-1–CD40L pathway becomes relevant only in the absence of CD40 molecules as is present in CD40 KO mice. Redundancy is an important and common occurrence in many biological systems because it provides fail-safe mechanisms and backups that are necessary for maintaining proper functioning of the host. The redundancy in IL-12 production pathway may become particularly important in individuals with genetic mutations in the CD40 gene. These individuals have been shown to have recessive form of hyper-IgM syndrome similar to the disease seen in individuals with mutations in the CD40L gene (46). The CD40L–Mac-1 pathway could be targeted in these individuals for therapeutic purposes.

In summary, our data show striking differences in the outcome of disease and immune response in L. major–infected CD40 and CD40L KO mice after rIL-12 treatment. These differences were related, in part, to alternative utilization of CD40L–Mac-1 pathway for continuous and sustained IL-12 production in infected CD40 KO mice. Thus, our studies reveal a critical and redundant role of Mac-1 in IL-12 production and resistance to L. major infection.

The authors have no financial conflicts of interest.