Interleukin-4-independent acceleration of cutaneous leishmaniasis in susceptible BALB/c mice following treatment with anti-CTLA4 antibody

BALB/c mice are susceptible to progressive infection with Leishmania major due to the preferential development of CD4(+) T cells that secrete Th2 cytokines. Although Th2 cell development and susceptibility are disrupted by blockade of CD86 function early in infection, CD28-deficient BALB/c mice remain susceptible to leishmaniasis. We therefore examined whether the alternative CD86 ligand, CTLA4, contributes to the expression of susceptibility. BALB/c mice treated for 2 weeks of infection with anti-CTLA4 monoclonal antibody developed more rapidly progressive disease than sham-treated mice, whereas normally resistant C57BL/6 mice were unaffected. The draining lymph node cells of anti-CTLA4-treated BALB/c mice produced up to sixfold more interleukin-4 (IL-4) and IL-13 than control mice in the first 2 weeks of infection, but IFN-gamma synthesis was reciprocally decreased. Anti-CTLA4 treatment of BALB/c mice pretreated with neutralizing anti-IL-4 antibody or genetically deficient in IL-4 also caused significant worsening of leishmaniasis. Exacerbation in IL-4 KO mice was associated with increased IL-13 and decreased gamma interferon (IFN-gamma) and inducible nitric oxide synthase (iNOS) mRNA expression in vivo. These data indicate that anti-CTLA4 antibody induced earlier and more-polarized Th2 responses in susceptible BALB/c mice infected with L. major. The mechanism of disease worsening was partially IL-4 independent, indicating that increased IL-13 and/or decreased IFN-gamma production may have disrupted nitric oxide-based microbicidal responses. We conclude that CTLA4 significantly modulates Th2 development in murine leishmaniasis and that the Th2-polarizing effects of anti-CTLA4 treatment result in IL-4-independent exacerbation of disease.     

Host genetic background determines whether IL-18 deficiency results in increased susceptibility or resistance to murine Leishmania major infection

Interleukin-18 (IL-18) plays an important role in innate and acquired immunity. IL-18 gene deficient (IL-18-/-) mice of the 129 x CD1 strain were reported to be more susceptible to Leishmania major infection than the wild-type mice. In contrast IL-18-/- mice of the C57BL/6 background were found to be as resistant as the wild-type (WT) mice. To resolve this discrepancy, IL-18 gene deficiency was introduced by backcrossing on to the highly susceptible BALB/c, or the moderately resistant DBA/1 backgrounds. Here we have demonstrated that BALB/c IL-18-/- mice were more resistant to L. major infection than WT BALB/c mice, whereas DBA/1 IL-18-/- mice were markedly more susceptible than their WT littermates. BALB/c IL-18-/- mice produced less IFNgamma and IL-4, whereas DBA/1 IL-18ko mice produced more IFNgamma and IL-4 than their respective WT controls. These result clearly demonstrate that the role of IL-18 in resistance or susceptibility to L. major is determined by host genetic background.     

Interleukin-4 (IL-4) and IL-10 collude in vaccine failure for novel exacerbatory antigens in murine Leishmania major infection

Leishmaniasis affects 12 million people but there are no vaccines in routine use. Recently, we used DNA vaccination in a susceptible BALB/c high-dose model of infection to screen 100 novel Leishmania major genes as vaccine candidates. In addition to finding novel protective antigens, we identified several antigens that reproducibly exacerbated disease. Here we examined the immune response to two of these antigens, lmd29 and 584C, that were originally identified in an expressed sequence tag cDNA sequencing project. We show that, in addition to exacerbating disease in susceptible BALB/c mice, these antigens retain a propensity to exacerbate disease in resistant C57BL/6 mice. This ability to exacerbate disease was lost when susceptible BALB/c mice were rendered resistant by disruption of the genes encoding interleukin-4 (IL-4) alone, IL-4/IL-13, or IL-4, IL-5, IL-9, and IL-13. Failure to exacerbate disease was associated with reduced IL-5 and IL-10 production in IL-4 knockout mice. Treatment of lmd29-vaccinated mice with anti-IL-10 receptor antibody prior to challenge infection converted exacerbation in wild-type BALB/c mice into highly significant antigen-specific protection. These studies demonstrate that some highly immunogenic antigens of L. major, while having an intrinsic capacity to exacerbate disease in the context of otherwise T helper 1-promoting DNA vaccine delivery, can be rendered protective by the removal of functional IL-10.     

Role of CD4(+)CD25(+) T regulatory cells in IL-2-induced vascular leak

T regulatory cells (CD4(+)CD25(+)) play an important role in the regulation of the immune response. However, little is known about the ability of T regulatory cells to regulate endothelial cell (EC) damage following activation of lymphocytes with IL-2. Therefore, in the current study, we examined the role of T regulatory cells and the subsequent T(h)1/T(h)2 bias in IL-2-mediated EC injury using the well-characterized C57BL/6 (T(h)1-biased) and BALB/c (T(h)2-biased) models. Following IL-2 treatment, BALB/c mice were less susceptible to IL-2-induced vascular leak syndrome (VLS) compared with C57BL/6 mice. Splenocytes from BALB/c mice displayed less cytotoxicity against ECs compared with those from C57BL/6 mice. Interestingly, BALB/c mice had significantly higher numbers of CD4(+)CD25(+) T regulatory cells, which proliferated more profoundly following IL-2 treatment, compared with CD4(+)CD25(+) T regulatory cells from C57BL/6 mice. In addition, T regulatory cells from naive BALB/c mice were more potent suppressors of anti-CD3 mAb-stimulated proliferation of T cells than similar cells from C57BL/6 mice. Depletion of T regulatory cells in both BALB/c and C57BL/6 mice led to a significant increase in IL-2-induced VLS. Together, the results from this study suggest that CD4(+)CD25(+) T regulatory cells play an important role in the regulation of IL-2-induced EC injury.     

The TGF-beta response to Leishmania chagasi in the absence of IL-12

Cure of leishmaniasis requires a type 1 immune response characterized by IFN-gamma production. Leishmania major infection leads to a type 2 response suppressing cure of susceptible BALB/c mice, and L. major causes an exacerbated type 2 response in mouse strains with a gene knockout (KO) such that they lack IL-12p40 (IL-12KO mice). In contrast, type 1 responses are inhibited by TGF-beta without Th2 cell expansion in BALB/c mice infected with L. chagasi. We questioned whether the type 2 or the TGF-beta response would dominate during L. chagasi infection of IL-12KO mice. C57BL/6 mice developed self-resolving L. chagasi infection with abundant IFN-gamma. In contrast, L. chagasi disease was exacerbated and IFN-gamma was low in IL-12KO mice. Total TGF-beta was significantly higher in IL-12KO than control C57BL/6 mice, but IL-4 and IL-10 levels were similar. TGF-beta was further augmented in IL-12/IFN-gamma double-KO mice. Thus, in contrast to L. major, the TGF-beta response was exacerbated whereas type 2 cells were not expanded during L. chagasi infection of IL-12KO mice. We conclude that L. chagasi has an inherent propensity to elicit a prominent TGF-beta response that either suppresses, or is suppressed by, a type 1 response. We propose this be termed a "type 3" immune response, which can antagonize a type 1 response.     

Lactobacillus rhamnosus Ingestion Promotes Innate Host Defense in an Enteric Parasitic Infection

Enteric parasite infections around the world are a huge economic burden and decrease the quality of life for many people. The use of beneficial bacteria has attracted attention for their potential therapeutic applications in various diseases. However, the effects of beneficial bacteria in enteric parasitic infections remain largely unexplored. We investigated the effects of ingestion of Lactobacillus rhamnosus (JB-1) in a model of enteric nematode (Trichuris muris) infection. C57BL/6 (resistant to infection), AKR (susceptible to infection), interleukin 10 (IL-10) knockout (KO), and mucin Muc2 KO mice were infected with T. muris and treated orally with probiotic JB-1 or medium. The mice were sacrificed on various days postinfection to examine goblet cells, epithelial cell proliferation, cytokines, and worm burdens. Treatment with JB-1 significantly enhanced worm expulsion in resistant C57BL/6 mice, and this was associated with increases in IL-10 levels, goblet cell numbers, and epithelial cell proliferation. Beneficial effects of JB-1 were absent in IL-10 KO and resistant mice treated with γ-irradiated bacteria. Live JB-1 treatment also expedited worm expulsion in Muc2 KO mice and, more importantly, in AKR mice (susceptible to infection). Injection of IL-10 directly into the colonic tissue of uninfected mice induced goblet cell hyperplasia. These findings demonstrate that JB-1 modulates goblet cell biology and promotes parasite expulsion via an IL-10-mediated pathway and provide novel insights into probiotic effects on innate defense in nematode infection.     

The innate immune responses of colonic epithelial cells to Trichuris muris are similar in mouse strains that develop a type 1 or type 2 adaptive immune response

Trichuris muris resides in intimate contact with its host, burrowing within cecal epithelial cells. However, whether the enterocyte itself responds innately to T. muris is unknown. This study investigated for the first time whether colonic intestinal epithelial cells (IEC) produce cytokines or chemokines following T. muris infection and whether divergence of the innate response could explain differentially polarized adaptive immune responses in resistant and susceptible mice. Increased expression of mRNA for the proinflammatory cytokines gamma interferon (IFN-gamma) and tumor necrosis factor and the chemokine CCL2 (MCP-1) were seen after infection of susceptible and resistant strains, with the only difference in expression being a delayed increase in CCL2 in BALB/c IEC. These increases were ablated in MyD88-/- mice, and NF-kappaB p65 was phosphorylated in response to T. muris excretory/secretory products in the epithelial cell line CMT-93, suggesting involvement of the MyD88-NF-kappaB signaling pathway in IEC cytokine expression. These data reveal that IEC respond innately to T. muris. However, the minor differences identified between resistant and susceptible mice are unlikely to underlie the subsequent development of a susceptible type 1 (IFN-gamma-dominated) or resistant type 2 (interleukin-4 [IL-4]/IL-13-dominated) adaptive immune response.     

Self-priming cell culture system for monitoring genetically-controlled spontaneous cytokine-producing ability in mice

To monitor genetically-controlled cytokine-producing ability in mice in vitro, we developed a high-density cell culture system, which is preferable for inducing CD4+ T cell-dependent self-priming responses without any antigenic stimulation. When BALB/c spleen cells were cultured at high density (over 1.0 x 10(7) cells/well) in 12-well culture plate, they spontaneously produced cytokines including IFN-gamma, IL-2, IL-3, IL-5 and IL-6. The spontaneous cytokine production in this self-priming cell culture (SPCC) system was totally dependent on MHC class II-restricted CD4+ T cells. It was demonstrated that Th2-type BALB/c background mice exhibited higher levels of spontaneous cytokine production in SPCC culture compared with Th1-type C57BL/6 mice. Moreover, using BALB/c x C57BL/6 F1 mice and B10D2 congenic mice, it was demonstrated that highly spontaneous cytokine-producing ability in BALB/c background is genetically dominant and it is controlled by non-MHC genes. Unexpectedly, BALB/c mice spontaneously produced higher levels of IL-2 and IFN-gamma than C57BL/6 mice. However, BALB/c mice revealed lower levels of CTL and NK cell-generation in SPCC system compared with C57BL/6 mice. These results suggested that genetically-controlled predisposition of BALB/c mice toward Th2 immunity appeared not to be derived from their poor IFN-gamma-producing ability but rather derived from their poor responsiveness to IFN-gamma.     

Leishmania major-infected murine langerhans cell-like dendritic cells from susceptible mice release IL-12 after infection and vaccinate against experimental cutaneous Leishmaniasis

Leishmania major-infected C57BL / 6 skin-dendritic cells (DC) are activated and release cytokines (including IL-12 p70), and likely initiate protective Th1 immunity in vivo (von Stebut, E. et al., J. Exp. Med. 188: 1547 - 1552). To characterize differences in DC function in mice that are genetically susceptible (BALB / c) and resistant (C57BL / 6) to cutaneous leishmaniasis, we analyzed the effects of L. major on Langerhans cell-like, fetal skin-derived DC (FSDDC) from both strains. BALB / c- and C57BL / 6-FSDDC ingested similar numbers of amastigotes, but did not ingest metacyclic promastigotes. Like C57BL / 6-FSDDC, infection of BALB / c-FSDDC led to up-regulation of MHC class I and II antigens, CD40, CD54, and CD86 within 18 h. L. major-induced BALB / c DC activation also led to the release of TNF-alpha, IL-6 and IL-12 p40 into 18-h supernatants. Infected BALB / c- and C57BL / 6-DC both released small amounts of IL-12 p70 within 72 h. Additional stimulation with IFN-gamma and / or anti-CD40 induced the release of more IL-12 p70 from infected BALB / c-DC than C57BL / 6-DC. Co-culture of control or infected BALB / c- and C57BL / 6-DC with naive syngeneic CD4(+) T cells and soluble anti-CD3 resulted in mixed, IFN-gamma-predominant responses after restimulation with immobilized anti-CD3. Finally, syngeneic L. major-infected DC effectively vaccinated BALB / c mice against cutaneous leishmaniasis. Genetic susceptibility to L. major that results from induction of Th2 predominant immune responses after infection does not appear to reflect failure of skin DC to internalize or respond to parasites, or the inability of BALB / c T cells to mount a Th1 response to DC-associated Leishmania antigens.     

C57BL/6 mice are more susceptible to antigen-induced pulmonary eosinophilia than BALB/c mice, irrespective of systemic T helper 1/T helper 2 responses

Inflammatory response differences between C57BL/6 and BALB/c mice following ovalbumin (OVA) sensitization and a single challenge were investigated. Serum immunoglobulin (Ig)E and IgG1 levels were higher in C57BL/6 mice than in BALB/c mice. In contrast, IgG2a levels in C57BL/6 mice were lower than in BALB/c mice. Furthermore, the number of eosinophils infiltrating into lungs in C57BL/6 mice was significantly higher than in BALB/c mice after OVA challenge. The levels of the T helper 2 (Th2)-type cytokines interleukin (IL)-4 and IL-5, generated in challenged C57BL/6 lung tissue, were also higher than in BALB/c lung tissue. The participation of IL-4 and IL-5 in the induction of eosinophil infiltration into the lungs was confirmed in both strains of mice by injection of anti-IL-4 and anti-IL-5 monoclonal antibodies (mAbs). However, following OVA stimulation, in vitro IL-4 and IL-5 production in splenocyte cultures from C57BL/6 mice was lower than in splenocyte cultures from BALB/c mice. These results indicate that C57BL/6 mice induce Th2-type responses in the lungs, while BALB/c mice induce T helper 1 (Th1)-type responses in the lungs, despite considerable production of IL-4 and IL-5 from splenocytes. Therefore, local immune responses are more important in the induction of allergic inflammation in the lungs and are different from systemic immune responses, which are thought to depend on genetic background.     

Trypanosoma rhodesiense infection in mice: sex dependence of resistance.

There is large variation in the survival of inbred mouse strains infected with Trypanosoma rhodesiense (EATRO 1886). Of those strains that survived for at least 22 days postinfection, female mice were markedly more resistant than male mice. The longer a strain survived, the greater was the difference in survival between male and female mice. Parasite counts were higher in male mice than in females, suggesting that the decreased resistance of males was due to their relative inability to control parasite growth. To determine the possible role of an X-linked resistance gene, resistant (C57BL/6) and susceptible (BALB/c) mice were mated, and their F1 progeny were infected with T. rhodesiense. There was no difference in the resistance between reciprocal F1 male mice (C57BL/6 X BALB/c versus BALB/c X C57BL/6), indicating that an X-linked gene does not account for the difference in resistance between susceptible and resistant mice.     

Differential expression of interleukin-5 mRNA+ cells and eosinophils in Nippostrongylus brasiliensis infection in resistant and susceptible strains of mice

Interleukin (IL)-5 is produced by both T cells and eosinophils and has been implicated in lymphocyte and eosinophil differentiation and maturation. The extent to which differences in IL-5 expression contribute to genetic variability in parasite immunity was investigated by comparing eosinophilia, IgE production, mastocytosis and IL-5 mRNA⁺ cells following Nippostrongylus brasiliensis infection of resistant (BALB/c) and susceptible (C57BL/6) mice. In uninfected mice, IL-5 mRNA⁺ cells detected by in situ hybridization were distributed throughout the lamina propria and crypt regions of the small intestine in both strains, but were 1.5-fold higher in BALB/c than in C57BL/6 mice. Following N. brasiliensis infection, the numbers of IL-5 mRNA⁺ cells in BALB/c mice continued to increase until day 11 post-infection at which time they were more than 4-fold more numerous than in uninfected control mice of the same strain. In C57BL/6 mice, IL-5 mRNA⁺ cells reached peak numbers on day 7 post-infection, only 1.5-fold higher than uninfected controls, but the numbers began to decline thereafter. At all time points after day 5, the numbers of IL-5 mRNA⁺ cells in the gut of C57BL/6 mice were significantly lower than BALB/c mice. The differences in numbers of IL-5 mRNA⁺ cells induced by infection in each strain of mice correlated with changes in blood and intestinal eosinophilia, mastocytosis and IgE production and was reflected in differences in worm expulsion and egg counts. Although numbers of intestinal IgA-containing cells increased in both strains after infection, there was no difference between strains except at day 11 when there were significantly higher numbers in BALB/c mice than in C57BL/6 mice. These results suggest that IL-5 is an important regulatory factor determining host immunity to parasite infection and that differential regulation of IL-5 expression explains in part the observed strain differences with respect to parasite resistance.     

CD4(+) and CD8(+) cells are key participants in the development of recurrent herpetic stromal keratitis in mice

Ocular herpes simplex virus (HSV) infection results in an immune-mediated inflammation of the corneal stroma known as herpetic stromal keratitis (HSK). Recurrent HSK is a common cause of virus-induced corneal blindness in humans. The role of CD4(+) and CD8(+) T cell subsets in the disease pathogenesis is ill defined and varies with the virus strain and host genetic background. To examine the contribution of T cell subsets to corneal disease, we studied the development of recurrent HSK in CD4 or CD8 gene knockout (KO) mice ocularly infected with HSV-1 McKrae strain. Following UV-B induced viral reactivation, corneal opacity in latently infected BALB/c (HSV sensitive) CD4 and CD8 KO mice was reduced compared to infected BALB/c mice with normal genotype. In contrast, opacity in C57BL/6 (HSV resistant) CD4 and CD8 KO latent mice did not differ from genetically normal latent mice. Virus-induced corneal opacity was not demonstrable in C57BL/6 CD4/CD8 double KO mice. Increased viral shedding, measured by reactivation rate, days shedding or viral titers, occurred in CD4 KO mice of both strains. Our findings indicate that both CD4(+) and CD8(+) cells play a role in the immunopathogenesis of recurrent HSK, and their role is dependent upon the host genetic profile.     

Yersinia enterocolitica infection in resistant and susceptible strains of mice.

We investigated natural resistance in mice to Yersinia enterocolitica, an enteric bacterial pathogen of humans, with a view to determine host genetic factors that are important in resistance. Most mouse strains studied (C3H/HeN, BALB/c, BALB.B, DBA/2, A, Swiss, and SWR) were highly susceptible to infection (50% lethal dose [LD50], 2 X 10(2) to 6 X 10(2) Y. enterocolitica administered intravenously [i.v.]). In contrast, C57BL/6 mice were highly resistant (LD50, 2 X 10(5) Y. enterocolitica administered i.v.). Resistance to i.v. Yersinia infection did not appear to be related to the Ity locus (which codes for resistance to Salmonella typhimurium and other pathogens) because Ityr mice (C3H/HeN, DBA/2, A, and SWR) were more susceptible to Y. enterocolitica than were Itys (C57BL/6) mice. In addition, because BALB.B mice (congenic to C57BL/6 mice at the H-2 locus) were susceptible, resistance was probably not H-2 linked. BALB/c X C57BL/6 F1 mice were intermediate in their resistance to Y. enterocolitica infection (LD50, 3 X 10(4) organisms administered i.v.), suggesting that resistance to Y. enterocolitica depends on a gene dosage affect or a resistance gene(s) interaction between susceptible and resistant parents. Further studies with C57BL/6 and BALB/c mice as prototype resistant and susceptible strains were undertaken. A time course study of Y. enterocolitica growth in various organs following i.v. infection revealed no strain difference in bacterial growth during the first 48 h of infection. Thereafter, however, C57BL/6 mice were capable of restricting Y. enterocolitica growth in all tissues (liver, lung, spleen, kidneys), whereas extensive bacterial proliferation occurred in BALB/c mice tissues. BALB/c mice were also more susceptible to oral Y. enterocolitica infection than were C57BL/6 mice, demonstrating increased mortality and greater numbers of bacteria in the Peyer's patches. Finally, whereas thymus-bearing C57BL/6 X BALB/c F1 mice were resistant to infection, athymic (nude) C57BL/6 X BALB/c F1 mice were susceptible. These studies provide a model to investigate natural immunity to enteric pathogens at mucosal surfaces, as well as provide the basis for clarifying the role of host genotype in Y. enterocolitica resistance.     

T cell independence of bleomycin-induced pulmonary fibrosis

The role of T cells and cytokines in bleomycin (BLM)-induced fibrosis was evaluated in susceptible and resistant strains of normal and SCID mice. Histology and hydroxyproline analysis showed that BLM induced pulmonary fibrosis in C57BL/6 and (C57BL/6 x BALB/c)F1 mice, whereas BALB/c mice were resistant to the disease. To test whether lymphocytes were required for the induction of BLM-induced pulmonary fibrosis, SCID mice were injected intratracheally with BLM and evaluated for the development of pulmonary inflammation and fibrosis. Similar morphological changes and increases in hydroxyproline were observed in both C57BL/6 SCID and (C57BL/6 x CB.17)F1 SCID animals compared to those seen in wild-type C57BL/6 and (C57BL/6 x BALB/c)F1 mice. In contrast, CB.17 SCID mice, which are genetically similar to BALB/c mice, were resistant to disease induction. Analysis of the cellular infiltrate in BLM-treated C57Bl/6 SCID mice confirmed a lack of T cells in the lungs of SCID mice and demonstrated a pronounced accumulation of eosinophils in areas of developing pulmonary fibrosis. NK cells were significantly elevated in untreated SCID mice and did not increase further after BLM treatment. Analysis of selected cytokines 1 day after initiation of BLM-induced pulmonary fibrosis indicated that the levels of TNF-alpha and IFN-gamma appeared to segregate with fibrosis in both the SCID and wild-type mice. The data demonstrate that T cells are not required for the induction of fibrosis by BLM and suggest that responses by non-lymphoid cells may be sufficient for the induction of fibrosis.     

Self-priming cell culture system for monitoring genetically-controlled spontaneous cytokine-producing ability in mice

To monitor genetically-controlled cytokine-producing ability in mice in vitro, we developed a high-density cell culture system, which is preferable for inducing CD4⁺ T cell-dependent self-priming responses without any antigenic stimulation. When BALB/c spleen cells were cultured at high density (over 1.0×10⁷ cells/well) in 12-well culture plate, they spontaneously produced cytokines including IFN-γ, IL-2, IL-3, IL-5 and IL-6. The spontaneous cytokine production in this self-priming cell culture (SPCC) system was totally dependent on MHC class II-restricted CD4⁺ T cells. It was demonstrated that Th2-type BALB/c background mice exhibited higher levels of spontaneous cytokine production in SPCC culture compared with Th1-type C57BL/6 mice. Moreover, using BALB/c×C57BL/6 F₁ mice and B10D2 congenic mice, it was demonstrated that highly spontaneous cytokine-producing ability in BALB/c background is genetically dominant and it is controlled by non-MHC genes. Unexpectedly, BALB/c mice spontaneously produced higher levels of IL-2 and IFN-γ than C57BL/6 mice. However, BALB/c mice revealed lower levels of CTL and NK cell-generation in SPCC system compared with C57BL/6 mice. These results suggested that genetically-controlled predisposition of BALB/c mice toward Th2 immunity appeared not to be derived from their poor IFN-γ-producing ability but rather derived from their poor responsiveness to IFN-γ.     

Dissociation of disease susceptibility, inflammation and cytokine profile in lmr1/2 congenic mice infected with Leishmania major

Severity of disease caused by Leishmania major depends on the genetics of the host. Early induction of T helper cell type 1 (Th1)-type responses in resistant C57BL/6 mice and T helper cell type 2 (Th2) in susceptible BALB/c mice is thought to determine cure or disease respectively. We have mapped three loci that confer susceptibility or resistance upon congenic mice on the C57BL/6 or BALB/c backgrounds. Here we examine the histopathology and production of interleukin 4 (IL-4) and interferon gamma (IFN-gamma) in the skin and draining lymph nodes in the congenic and parental mice. We show an evolving granuloma with a staged infiltration of inflammatory cells, but no difference between the groups. As an indication of an early-polarised Th1/Th2 response we measured IFN-gamma and IL-4 in the lymph nodes and found no difference between any of the mice during the first 48 h. During infection, the level of IL-4 correlated with the lesion size, indicating that IL-4 reflects the disease severity rather than controls it. Considering this effect, B6.C(lmr1,lmr2) mice had similar cytokine levels to the parental C57BL/6 mice despite increased susceptibility and C.B6(lmr1,lmr2) were similar to BALB/c despite increased resistance. We conclude that the lmr loci affect disease severity by a mechanism independent of conventional helper T-cell responses.