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Infect Immun. 2009 December; 77(12): 5668–5675.
Published online 2009 September 21. doi:  10.1128/IAI.00802-08
PMCID: PMC2786468

Complement Receptor 3 Deficiency Influences Lesion Progression during Leishmania major Infection in BALB/c Mice[down-pointing small open triangle]

Abstract

Leishmania major is an obligately intracellular protozoan parasite that causes cutaneous leishmaniasis. Like numerous intracellular pathogens, Leishmania exploits cell surface receptors as a means of entry into host cells. Complement receptor 3 (CR3; also called CD11b/CD18), a β2 integrin on phagocytic cells, is one such receptor. Ligation of CR3 has been shown to inhibit the production of interleukin-12, the cytokine that is pivotal in establishing the cell-mediated response necessary to combat intracellular infection. Here we investigate the role that CR3 plays in the establishment and progression of cutaneous leishmaniaisis in vivo. Dermal lesions of wild-type BALB/c mice are characteristically progressive and lead to extensive tissue necrosis coupled with elevated parasite burdens; CD11b-deficient BALB/c mice, however, demonstrate an intermediate phenotype characterized by chronic lesions and a reduced incidence of tissue damage. Infection followed by a reinfection challenge indicates that both susceptible (BALB/c) and resistant (C57BL/6) mice, regardless of CD11b status, develop resistance to L. major. In addition, CD11b does not bias the T helper cytokine response to L. major infection. Our results further indicate that CD11b is not necessary for disease resolution in resistant mice; rather, this protein appears to play a minor role in susceptibility.

Leishmania species are a group of intracellular protozoan parasites that infect cells of the monocyte/macrophage lineage. These parasites cause a range of clinical manifestations, from localized, self-limiting cutaneous lesions to systemic fatal infections. Approximately 350 million people are at risk of infection worldwide (3), and an estimated 2 million new infections occur annually (16).

Leishmania entry into host cells is receptor mediated. Leishmania parasites have been shown to engage Fc receptors (FcR) (62), mannose receptor (8), Toll-like receptors 2, 3 (24), and 4 (37), and complement receptor 3 (CR3; also called Mac-1 or αMβ2) (46); however, the interactions of Leishmania parasites with CR3 have been the best characterized. CR3 is a versatile leukocyte-associated receptor with a number of endogenous and pathogen-associated ligands; as a result, this protein has multiple functions, playing roles in immunity, adhesion, and cell migration (21). Such versatility is a reflection of the structure of CR3 as a heterodimer of CD11b and CD18. Most ligands interact with the CD11b chain lectin and I domains, which recognize mainly pathogen-associated molecules (21) and endogenous ligands (33), respectively. The ligand binding promiscuity of CR3 includes extracellular matrix proteins (63), ICAM-1 (40), and bacterial lipopolysaccharide (LPS) (42). The best-defined function of CR3 is its role as the receptor for C3bi, a complement component protein (35). Interestingly, the predominant Leishmania surface molecule lipophosphoglycan is readily opsonized by complement (17) and binds to CR3 directly (58). Although CR3 is present on the very cells that are meant to control Leishmania infection, interaction with this receptor is thought to allow a silent means of entry for the parasite.

Leishmania parasites actively inhibit host immune responses to make their intracellular environments more hospitable. Leishmania-infected macrophages (MP) exhibit decreased gamma interferon (IFN-γ)-mediated major histocompatibility complex class II expression (38), defective IFN-γ signaling (50), lack of an oxidative burst (26, 49), and inhibition of interleukin-12 (IL-12) production (38). Despite the role of CR3 in immunity, Leishmania species purportedly utilize CR3 to gain entry into host cells without activating the production of reactive oxygen intermediates (25, 48). CR3 ligation, even in the absence of Leishmania infection, inhibits IL-12 expression (41), invoking the intriguing model that Leishmania parasites enter host cells via CR3-mediated phagocytosis to evade host immune responses and thus establish infection.

The role of CR3 during cutaneous leishmaniasis has been investigated previously using a CD18-deficient (CD18 KO [knockout]) 129SV × C57BL/6 murine model of infection. This study demonstrated that uptake of serum-opsonized Leishmania inhibited IL-12 production in wild-type (WT) MP but not in CD18 KO MP. Paradoxically, however, CD18 KO mice harbored more parasites than WT mice and exhibited parasite dissemination. In this particular case, the defect in parasite clearance was due to the additional absence of other CD18-containing β2 integrins, LFA-1 and CR4, in the T-cell compartment (50); therefore, these studies do not specifically address a role for CR3.

Here we have examined the role that CR3 plays in the establishment and progression of L. major infection by using a murine model of susceptible and resistant WT and CD11b-deficient (CD11b KO) mice. Our data indicate that in the absence of CD11b, BALB/c mice exhibit increased resistance to L. major infection.

MATERIALS AND METHODS

Mice and parasites.

WT BALB/c, C57BL/6, and CBySmn.CB17 (BALB/c SCID) mice were purchased from Jackson Labs (Bar Harbor, ME). CD11b-null mice, generated by disrupting the exon encoding the translational initiation codon with a neomycin gene cassette, were the generous gift of Tanya Mayadas (Brigham and Women's Hospital and Harvard Medical School) (15). These mice were originally generated on a C57BL/6 × 129SV background and were backcrossed 8 generations to both C57BL/6 and BALB/c strains (32). A CR3 WT line on each background was generated from a CD11b heterozygote cross at the University of Notre Dame. All animals were housed at the University of Notre Dame's Friemann Life Sciences Center according to IACUC standards. Leishmania major strain Friedlin V1 (MHOM/IL/80/Friedlin) parasites were cultured at 26°C without CO2 in complete medium 199 (M199C) supplemented with 20% heat-inactivated fetal bovine serum (HyClone, Logan UT), 100 U/ml of penicillin, 100 μg/ml of streptomycin, 2 mM l-glutamine (Cellgro Technologies, Manassas, VA), 40 mM HEPES, 0.1 mM adenine, 5 μg/ml hemin in 50% triethanolamine, 1 mg/ml biotin, and 2.2 mg/ml sodium bicarbonate. Infective-stage metacyclic promastigotes were enriched from 5-day-old stationary-phase cultures via a Ficoll density gradient as previously described (54). Briefly, parasites were pelleted and resuspended in Dulbecco's modified Eagle medium; then they were centrifuged over a gradient composed of 2 ml of 10% Ficoll over 2 ml of 20% Ficoll, after which the top two layers containing the infective-stage parasites were collected, washed, and counted on a hemocytometer. For in vitro infection, metacyclic parasites were opsonized with 5% normal mouse serum in Hanks buffered salt solution containing 0.15 mM Ca2+ and 1.0 mM Mg2+ for 30 min at 37°C; then they were washed and resuspended in RPMI 1640 medium (RPMI-C) (CellGro Technologies, Manassas, VA) containing 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Amastigotes were isolated from footpad lesions of BALB/c WT and SCID mice as described previously (52).

In vivo infection.

Six- to 8-week-old female mice were infected intradermally in the ears with either a low dose (1 × 103) or a high dose (1 × 105) of L. major parasites. Infection of the ear dermis was performed in 20 μl of sterile phosphate-buffered saline (PBS). Developing lesions were monitored over the course of several weeks by measuring the diameter with electronic calipers. For the experiments for which results are shown in Fig. Fig.2,2, mice were infected in the right ear with 1 × 105 parasites, and lesions were monitored in the manner described above. Two weeks after the C57BL/6 lesions had healed, mice were challenged in the left ear with 1 × 105 parasites, and subsequent lesions on both ears were recorded.

FIG. 2.
The murine genetic background does not dictate or preclude the development of resistance to Leishmania. (A) Female mice were infected with 105 L. major parasites in the right ear, and the developing lesions were monitored. Mean lesion diameters ± ...

Parasite quantification.

At various time points throughout infection, random mice were euthanized with CO2, and the infected ears were removed to 70% ethanol. Under sterile conditions, the dermal layers of each ear were separated and incubated in RPMI-C containing 1 mg/ml collagenase (Gibco/Invitrogen, Carlsbad, CA) for 1.5 h at 37°C. Ear tissue was homogenized by clipping 100 times with sterile surgical scissors. Subsequently, the ear homogenate was filtered with 70-μm-pore size Cell Strainers (BD Falcon, Franklin Lakes, NJ), and parasites were pelleted out of the filtrate by centrifugation at 1,250 × g. The resulting parasites were resuspended in 200 μl of M199C and were serially diluted 1:2 in 96-well plates containing Novy-Nicolle-McNeal (NNN) blood agar medium as described previously (1). Plates were incubated at ambient temperature for 7 days, after which wells were visually analyzed for the presence of parasites. Relative numbers of parasites per ear were determined by calculating back based on the number of dilutions performed.

Cell recruitment.

BALB/c WT and CD11b KO mice were infected intradermally in each ear as described above. Mice were euthanized at various time points postinfection (p.i.), and after collagenase incubation, the ears were homogenized using a Medimachine (Becton Dickinson, Franklin Lakes, NJ) tissue grinder system. The homogenate was filtered with 70-μm-pore-size syringe-fitting Filcons (BD Biosciences, Franklin Lakes, NJ). Isolated cells were pelleted by centrifugation and stained for fluorescence-activated cell sorter analysis. Cells were stained with fluorophore-conjugated antibodies used at 1:100. Analysis was carried out on a Beckman Coulter Epics XL flow cytometer. Leukocytes were identified by gating on total cells for granularity and the presence of phycoerythrin-labeled CD45 (BD Pharmingen, Franklin Lakes, NJ). Neutrophils and macrophages were identified by the presence of Ly-6 (BD Pharmingen, Franklin Lakes, NJ) and F4/80 (Caltag/Invitrogen, Carlsbad, CA), respectively, among the cell population that was CD45 positive. Cell counts were determined as the number of positive cells within the population of harvested cells from ear tissue.

Phagocytosis/clearance assay.

Bone marrow was harvested by euthanizing mice with CO2 and flushing femurs with complete RPMI-C. Bone marrow MP (BMMP) were cultured on coverslips in L929 cell supernatant-conditioned RPMI-C medium for 7 days. Day 7 cells were infected 10:1 with L. major amastigotes for 0.5 h, 1 h, 4 h, or 24 h upon adherence on day 7. To determine BMMP competence for amastigote clearance, extracellular parasites were washed off after 16 h of infection, and cells were subsequently stimulated with 1,000 U/ml IFN-γ (Peprotech, Rock Hill, NJ) for 8 h, followed by 1 μg/ml LPS (Sigma-Aldrich, St. Louis, MO) for 24 h or 48 h. Coverslips were stained with Diff-Quick, mounted on slides, and counted for parasite burdens.

IgG subclass ELISA.

Mice were infected in the ear dermis as outlined above. At weeks 2, 4, 6, 8, and 10 p.i., mice were eye bled for serum collection. Uninfected mice served as a control group. Blood was allowed to coagulate at room temperature for 2 h, and samples were centrifuged at 13,000 × g for 10 min to separate serum from clots. Approximately 50 μl of serum was isolated from each mouse and stored at −80°C until use. Serum was diluted at 1:100 in PBS and analyzed by an enzyme-linked immunosorbent assay (ELISA) using soluble Leishmania antigen as a capture antigen. Antigen-specific total immunoglobulin G (IgG), IgG1, IgG2a, IgG2b, and IgG3 were assessed, according to the manufacturer's instructions, in duplicate (Southern Biotech, Birmingham, AL). To enable comparisons between ELISA plates, all samples were normalized to a reference sample analyzed on the same plate using the following formula: (average of reference sample values over all plates)/(reference sample value on the plate) × (experimental sample value). The reference sample consisted of a pool of serum samples collected from all the time points analyzed.

Bone marrow-derived dendritic cell (DC) culture and infection.

Six- to 8-week-old female mice were euthanized via CO2, and femurs were isolated. Bone marrow was flushed with RPMI-C, and erythrocytes were lysed in ACK buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2 EDTA). Cells were cultured at 2 × 105/ml in granulocyte-macrophage colony-stimulating factor (Peprotech, Rocky Hill, NJ) at 20 ng/ml for 12 days and were used on day 13. Cells were collected, plated in 6-well plates, and infected 10:1 with opsonized L. major for 16 h.

LN restimulation.

Mice were euthanized at day 2, week 2, and week 8 (at the earliest onset of visible necrosis) p.i., and submandibular lymph nodes (LNs) were collected into sterile PBS from four mice per group and pooled. LNs were homogenized on 70-μm-pore-size Cell Strainers (BD Falcon, Franklin Lakes, NJ) by disruption with the end of a syringe barrel plunger. Cells were washed with RPMI-C, pelleted, and counted, after which they were aliquoted at 106 cells in 200 μl to round-bottom 96-well plates. LN cell suspensions were stimulated with L. major-infected isogenic bone marrow-derived DCs at a DC/LN cell ratio of 1:5. Concanavalin A (Sigma-Aldrich, St. Louis, MO) was used as a control at 1 μg/ml. Cells were incubated at 37°C for 5 days, after which they were pelleted, and supernatants were frozen down for analysis on the Luminex 100 IS system (Luminex Corp., Austin, TX) with Lincoplex multiplex assay kits (Linco Research/Millipore, Billerica, MA). Data were analyzed with BeadLyte software.

Statistical analysis.

For statistical analysis of lesion sizes, analysis of variance between WT and CD11b-KO strains (performed for BALB/c and C57BL/6 separately) was utilized with a subsequent Bonferroni posttest to determine at what time points the strains were different. A two-tailed Mann-Whitney test was used to determine differences in parasite numbers. Student's t test was utilized for in vitro phagocytosis assays. All statistical analyses were performed using GraphPad Prism software, version 5.0. In all cases, a P value of <0.05 was considered statistically significant.

RESULTS

CR3-deficient BALB/c mice demonstrate attenuated Leishmania major infection.

BALB/c mice develop progressive dermal lesions in response to L. major infection, while C57BL/6 mice develop small, localized, self-healing lesions. We investigated the role of CR3, a known receptor for parasite entry into host cells, on the development and progression of dermal lesions (Fig. (Fig.1A).1A). Infected C57BL/6 mice exhibit similar, resistant courses of lesion development regardless of the availability of CR3. BALB/c WT mice exhibit the typical progressive lesion development; CD11b-deficient BALB/c mice present similar lesion kinetics (i.e., lesions develop at approximately the same time p.i.), but by week 8, their lesions are smaller, and eventually a proportion decrease in size (Fig. (Fig.1B)1B) and remain chronic (data not shown). Although lesions are attenuated in BALB/c CD11b KO mice, parasite quantification indicates no difference in parasite numbers at the lesion sites of WT and CD11b KO mice (Fig. (Fig.1C).1C). Interestingly, BALB/c WT mice also developed tissue necrosis sooner and more profusely than their CR3-deficient counterparts (data not shown). By 13 weeks p.i., very few CD11b KO mice had developed dermal necrosis, while approximately 45% of WT ears had some level of tissue damage, ranging from a notch in the ear tissue to complete destruction of the ear dermis. Taken together, these data indicate that CD11b-deficient BALB/c mice exhibit an intermediate phenotype of L. major lesion progression.

FIG. 1.
Lesion sizes and parasite burdens in WT and CD11b-deficient mice infected intradermally in the ear with 103 L. major parasites. (A) Lesion sizes in BALB/c and C57BL/6 mice were measured over the course of 12 weeks. Mean lesion diameters ± standard ...

Resistance to secondary L. major challenge is intact in CD11b-deficient mice.

The Th2 immune response characteristic of Leishmania-infected BALB/c mice is not sufficient to clear an initial infection. Rather, the parasite load continues to increase, along with the lesion size, to the point of tissue damage and necrosis. Because BALB/c CD11b KO mice exhibit increased resistance to Leishmania infection in terms of pathology, we tested to see if this translated into resistance to reinfection (Fig. (Fig.2).2). Previous reports have indicated that low-dose infections in BALB/c mice can induce protection upon secondary Leishmania major challenge (10); therefore, we switched to a high-dose (105) infection model. Mice were initially infected in their left ears, and lesion sizes were monitored as for Fig. Fig.1;1; upon healing of C57BL/6 mice, all mice were reinfected in the right ear (week 14). Primary lesions developed and resolved as expected (Fig. (Fig.2A).2A). All secondary lesions exhibited by C57BL/6 mice were transient and healed completely over the course of a few weeks (Fig. (Fig.2B).2B). In spite of uncontrolled lesion development at the initial infection site in BALB/c WT mice, lesions at the challenge site were attenuated, resulting in small, chronic lesions, a mechanism that was not affected by the absence of CD11b (Fig. (Fig.2B).2B). Healing of challenge lesions was observed in 25% of WT and 36% of CD11b KO mice by the end of the experiment. Interestingly, we did not observe obvious reactivation of C57BL/6 lesions, contrary to previous reports (44). Furthermore, the healing and progression of the original lesions did not accelerate. These data indicate that the type of initial response (progressive, attenuated, healing) neither dictates nor precludes the development of resistance to a secondary challenge.

CD11b KO and WT BMMP host cells phagocytose amastigote parasites with equal efficiency.

Although no statistically significant differences in lesion parasite numbers were detected (Fig. (Fig.1C),1C), mean parasite numbers were lower in BALB/c CD11b KO mice than in WT mice at weeks 4, 8, and 13. To assess if these lower parasite burdens were due to a partial defect in the ability of MP to phagocytose parasites, we infected BMMP in vitro with lesion-derived amastigotes. The data demonstrate that WT and CD11b KO BMMP phagocytose amastigotes isolated from WT mice equivalently (Fig. (Fig.3A).3A). Amastigotes isolated from WT mice are opsonized with both IgG and complement components (29, 47). To assess the role of CD11b in the uptake of parasites in the absence of parasite-specific IgG, we infected BMMP with amastigotes isolated from SCID mice; again, no difference in parasite uptake between WT and CD11b KO BMMP was detected. CR3 engagement has been implicated in inhibition of IFN-γ signaling (41, 56) and in evasion of the production of reactive oxygen intermediates (6), suggesting that the utilization of CR3 by Leishmania parasites allows them to persist within their host cells. To determine if CR3 engagement by Leishmania inhibits activation for intracellular killing, infected cells were stimulated and parasite burdens assessed. Cells, WT or CD11b KO, activated with IFN-γ plus LPS stimulation exhibited dramatic decreases in parasite loads by 48 h poststimulation (Fig. (Fig.3B).3B). Taken together, these data indicate that parasites can enter BMMP regardless of CR3 and that these cells can be activated for parasite killing.

FIG. 3.
BMMP from WT and CD11b-deficient BALB/c mice are competent to phagocytose and clear intracellular L. major parasites. (A) BMMP were infected with lesion-derived amastigotes isolated from WT or SCID mice at an MOI of 10:1 for the indicated times. Graphs ...

There is no difference in IgG1 or IgG2a antibody secretion between BALB/c WT and CD11b KO mice.

The production of IgG1 and IgG2a is considered indicative of Th2- and Th1-biased immune responses, respectively. To test if infected CR3-deficient mice were skewed toward a resistant Th1 response, the levels of serum IgG subclasses of antibodies were assayed. Serum samples collected at various time points post-L. major infection were analyzed by ELISA for the production of total IgG and isotypes IgG1, IgG2a, IgG2b, and IgG3. The data indicate that there is no difference in either IgG1 or IgG2a production in L. major-infected BALB/c mice (Fig. 4A and B). Levels of both antibody subtypes began to increase about 4 weeks p.i. and continued to rise up to week 10, the last time point assayed, which was shortly after WT and KO BALB/c mice developed divergent phenotypes. Additionally, both experimental groups produced approximately the same amounts of total IgG, IgG2b, and IgG3 (data not shown).

FIG. 4.
BALB/c WT and BALB/c CD11b KO mice exhibit no difference in IgG1 (Th2) and IgG2a (Th1) production. Female mice were infected with 105 L. major parasites, and serum samples were collected at the indicated times. Samples were analyzed by ELISA for IgG subclasses ...

CR3-deficient lesions demonstrate a greater initial MP and neutrophil influx.

CR3 plays an important role during immune responses but is also important for cell chemotaxis and extravasation. Therefore, we analyzed ear tissue to identify cellular components and to determine if CR3 deficiency may modulate cell recruitment into the infected lesion. Ear tissue was analyzed by flow cytometry to characterize the cellular infiltrate at various time points p.i. (Fig. (Fig.5).5). The leukocyte population was identified by the presence of CD45, and the F4/80+ (macrophages) and Ly6+ (neutrophil) populations were identified as subsets. Approximately 20 to 40% of leukocytes present in uninfected tissue are F4/80+ macrophages. The percentage of F4/80+ cells decreases 1 day p.i. in WT mice, and by day 2 p.i., 40% of leukocytes in both WT and CR3-deficient tissues are F4/80+. Neutrophils are present at low levels in uninfected tissues of both genotypes but infiltrate more rapidly into CR3-deficient mice; by day 1, 60% of leukocytes are Ly-6+ in CD11b KO mice, while WT lesions do not reach this level until a day later.

FIG. 5.
CD11b-deficient lesions show increased influx of MP and neutrophils early in infection. Female mice were infected with 105 L. major parasites, and ear tissue was harvested at various time points. Cell infiltrates were pooled from each time point and examined ...

WT and CR3-deficient mice display similar profiles of Th1 and Th2 cytokine production.

We further investigated the role of CR3 in Leishmania infection by investigating cytokine-mediated recall responses of infected mice. L. major-infected syngeneic DCs were coincubated with LN cells, and supernatants were assayed for IFN-γ, IL-4, and IL-10. Our data indicate that IFN-γ is produced as early as day 2 p.i. in both WT and KO mice and that its levels in the two strains remain equal and increase up to week 9 p.i. (Fig. (Fig.6A).6A). IL-4 and IL-10 are not substantially upregulated until week 2 p.i. and are produced equally by WT and KO LN cells. IL-4 levels increase gradually through week 9 (Fig. (Fig.6B),6B), while IL-10 levels increase substantially at that time point (Fig. (Fig.6C).6C). These data indicate that CR3 plays a minimal role in cytokine-mediated immune responses to L. major infection and that the increased resistance to infection in CR3-deficient mice is not the result of an increase in Th1-type cytokines.

FIG. 6.
BALB/c CD11b-deficient and WT mice produce similar levels of Th1 and Th2 cytokines. Memory recall responses of infected mice were analyzed by a Luminex multiplex assay. Infected DC from syngeneic mice were coincubated with lymph node cells collected from ...

DISCUSSION

CR3 has been shown to play a role in infection with a number of pathogens, including Toxoplasma gondii (34), Neisseria gonorrheae (20), Coxiella burnetii (11), Cryptococcus neoformans (57), Mycobacterium species (32, 43), Porphyromonas gingivalis (30), and possibly Leishmania species (53). Although CR3 is a prominent receptor on professional phagocytes, CR3 also is thought to be a common port of entry for pathogens, because CR3 ligation inhibits IL-12 production (41) and bypasses the production of reactive antimicrobial molecules (13). By utilizing CR3, pathogens are able to infect their host cells in a silent manner and avoid activating immune responses.

Our studies have examined the role that CR3 plays in Leishmania major infection. In vivo dermal infection with infective-stage metacyclic promastigotes indicates that BALB/c CD11b KO mice are more resistant to L. major infection in terms of pathology than their WT counterparts, as evidenced by their smaller lesions, lower incidence and extent of ulceration, and delayed onset of necrosis. While lesion progression is altered in BALB/c CD11b KO mice, parasite burdens appear to be equivalent to those in WT mice. This disconnection between parasite load and lesion growth is achieved because much of the inflammation and damage that develop during cutaneous leishmaniasis are caused by the immune response, not directly by the parasite (51). Taken together, our data indicate that lack of CR3 alters the course of Leishmania infection such that susceptible mice display an intermediate phenotype of disease presentation.

CR3 does not play a significant role in memory and the development of immunity to Leishmania or in the ability of the host cell BMMP to take up the parasite. Infected mice challenged with a secondary infection at a distal site developed small, localized lesions with little or no ulceration. The onset of immunity is not affected by the absence of CD11b on either genetic background. It is noteworthy that neither the development nor the healing of the primary lesions of any of the strains tested was accelerated during the time course of infection.

A healing BALB/c phenotype concomitant with immunity to reinfection has previously been reported for mice infected with a low dose of Leishmania parasites (10). Healing BALB/c phenotypes, while unusual, have been described in the literature in terms of their requirements. At the onset of a primary infection with L. major, parasites multiply at the site of infection, and lymphocytes from infected mice produce IFN-γ, IL-10, IL-4, and IL-13, clearly indicating no obvious bias toward Th1 or Th2 immunity. Mice infected with very low doses of parasites never develop cutaneous lesions, and these mice do not become immune to subsequent infection, indicating that lesion development plays an important role in the acquisition by BALB/c mice of what is termed “concomitant immunity” (14).

Several groups have examined the relationship between parasite dose and subsequent immune response, with mixed results. A number of reports state that infection with high doses of Leishmania lead to a nonprotective Th2 response in BALB/c mice, while low-dose infections skew the immune response to Th1 (14, 39, 59). Although there are data echoing both phenotypes in resistant C57BL/6 mice as well (45, 60), there is no clear indication that the Leishmania dose will necessarily polarize immunity one way or the other. We should, however, bear in mind that such skewing has also been noted in other, non-Leishmania systems (9, 28, 31).

Activated BMMP clear parasites soon after IFN-γ and LPS stimulation, leading to a precipitous drop in the parasite burden to nearly undetectable levels by 48 h poststimulation; this drop is not dependent on CR3, since both WT and CD11b-deficient cells exhibit the same phenomenon. While these data do not explain the phenotypic differences detected between WT and KO mice in vivo, they are not surprising for a number of reasons. First, MP are not the only phagocytes present in the skin; DCs (36) and neutrophils (12, 27) have been reported to harbor Leishmania parasites. Furthermore, CR3 is only one of a number of receptors that have been reported to bind Leishmania parasites and play a role in their uptake. These other receptors, such as FcR, mannose receptor, and Toll-like receptor 4, may act as part of a compensatory mechanism so that parasites are internalized at levels comparable to those for WT mice. Clearly, while MP and CR3 are involved in the in vivo development of L. major-mediated disease, they are not exclusive players.

Memory and recall responses of LNs to L. major-infected DCs further support the notion that CR3 plays only a small role in immunity to leishmaniasis. Our analyses of cytokine secretion demonstrated that CD11b-deficient mice produce levels of IFN-γ, IL-4, and IL-10 in the draining LNs similar to levels for WT mice. Interestingly, although BALB/c WT mice develop standard progressive lesions characteristic of L. major infection, they also produce IFN-γ early in infection. IFN-γ is detected as early as day 2 p.i., and its levels increase over the 9-week course of infection. IL-10 and IL-4, characteristic of the Th2 response, are not clearly upregulated until 2 weeks p.i. regardless of the presence of CD11b. Despite the purported mutual exclusivity of the Th1 and Th2 cytokine responses, it is now becoming clear that T helper profiles alone do not govern the outcome of cutaneous leishmaniasis. It has recently come to light that wound healing makes an equally important contribution to the control and subsequent clearance of Leishmania parasites, since cytokine patterns are often not consistent with the severity of disease witnessed in vivo (5, 22). Mice congenic for genes associated with resistance to Leishmania heal faster, in part as the result of more orderly collagen fiber repair and deposition (4). It must be stressed that this is a noteworthy phenomenon, because the genes regulating these responses, termed “leishmania host reponse loci” (lmr1 to lmr3), function independently of the Th1/Th2 cytokine phenotype of the host in question (23), leading to the belief that the type of T-cell response does not necessarily dictate whether a host will cure a Leishmania infection and that the process of wound healing is a phenomenon separate from parasite control and clearance.

Our analyses also involved an examination of cell types present at the site of infection. Neutrophils are generally more prevalent in CD11b KO lesions than in WT lesions early during infection. As previously reported (7), neutrophil numbers remained high in BALB/c WT infiltrates. Interestingly, phenotypically resistant mice (e.g., C57BL/6 mice) exhibit transient neutrophil accumulation (55), similar to what we detected in CD11b-deficient BALB/c lesions (Fig. (Fig.5).5). In contrast to what was observed in ear tissue, inflammatory neutrophils have a delay in apoptosis and accumulate in the peritoneal cavities of CD11b-deficient mice. This deregulation of apoptosis and neutrophil accumulation is attributed to a decrease in oxygen radical generation (15). While we have not assessed reactive oxygen species in neutrophils, there is not a defect in WT or KO MP, as evidenced by the fact that the killing mechanisms in activated cells remained intact despite a lack of CR3.

As β2 integrins, CR3 and other CD18-containing molecules are involved in cell attachment and motility. Previous reports indicate that CR3 is in fact involved in cell attachment. CD11b KO leukocytes have a higher average rolling velocity than WT leukocytes (18), because they are not as capable of attaching to endothelia via ICAM-1 (19, 61). CR3 also can bind fibrinogen, and this interaction has been shown to promote leukocyte adhesion as well (2). Presumably, in the absence of CR3, we might detect a defect in cellular extravasation to sites of infection. However, our data revealed the opposite phenomenon, an increase in levels of CD45+ MP and neutrophils soon after infection.

In summary, our data indicate that L. major infection is less severe in CD11b-deficient mice than in BALB/c WT animals. Because infective-stage parasites are still able to establish themselves in host cells, they certainly are utilizing other receptors as mechanisms of entry. While a lack of CD11b does not clearly bias a normally Th2-type immune response to a Th1-type immune response, it does correlate with increased levels of MP and neutrophil infiltration early in infection. CD11b also has little if any effect on the development of cell-mediated memory, since there is no difference between WT and KO animals after challenge infection. Clearly, CD11b has only a modest effect on L. major infection in vivo.

Acknowledgments

We are grateful to Tanya Mayadas (Brigham and Women's Hospital and Harvard Medical School) for the use of CD11b-KO mice. We thank the Freimann Life Science Center at the University of Notre Dame for excellent animal care.

This work was supported by grants to M.A.M. from the National Institutes of Health (NIH) (RO1AI056242) and the American Heart Association (0435333Z). C.R.C. was supported by a training grant from the NIH (T32AI07030).

Notes

Editor: J. F. Urban, Jr.

Footnotes

[down-pointing small open triangle]Published ahead of print on 21 September 2009.

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