IFN-gamma production by intrinsic renal cells and bone marrow-derived cells is required for full expression of crescentic glomerulonephritis in mice

The contribution of IFN-gamma from bone marrow (BM) and non-BM-derived cells to glomerular and cutaneous delayed-type hypersensitivity (DTH) was studied in mice. Chimeric IFN-gamma mice (IFN-gamma(+/+) BM chimera), in which IFN-gamma production was restricted to BM-derived cells, were created by transplanting normal C57BL/6 (wild-type (WT)) BM into irradiated IFN-gamma-deficient mice. BM IFN-gamma-deficient chimeric mice (IFN-gamma(-/-) BM chimera) were created by transplanting WT mice with IFN-gamma-deficient BM. WT and sham chimeric mice (WT mice transplanted with WT BM) developed crescentic glomerulonephritis (GN) with features of DTH (including glomerular T cell and macrophage infiltration) in response to an Ag planted in their glomeruli and skin DTH following subdermal Ag challenge. IFN-gamma-deficient mice showed significant protection from crescentic GN and reduced cutaneous DTH. IFN-gamma(+/+) BM chimeric and IFN-gamma(-/-) BM chimeric mice showed similar attenuation of crescentic GN as IFN-gamma-deficient mice, whereas cutaneous DTH was reduced only in IFN-gamma(-/-) BM chimeras. In crescentic GN, IFN-gamma was expressed by tubular cells and occasional glomerular cells and was colocalized with infiltrating CD8(+) T cells, but not with CD4(+) T cells or macrophages. Renal MHC class II expression was reduced in IFN-gamma(+/+) BM chimeric mice and was more severely reduced in IFN-gamma-deficient mice and IFN-gamma(-/-) BM chimeric mice. These studies show that IFN-gamma expression by both BM-derived cells and intrinsic renal cells is required for the development of crescentic GN, but IFN-gamma production by resident cells is not essential for the development of cutaneous DTH.     

An IL-12-independent role for CD40-CD154 in mediating effector responses: studies in cell-mediated glomerulonephritis and dermal delayed-type hypersensitivity

Crescentic glomerulonephritis (GN) results from IL-12-driven Th1-directed cell-mediated responses (akin to delayed-type hypersensitivity (DTH)) directed against glomerular Ags. CD40-CD154 interactions are critical for IL-12 production and Th1 polarization of immune responses. Crescentic anti-glomerular basement membrane GN was induced in C57BL/6 (wild-type (WT)) mice (sensitized to sheep globulin) by planting this Ag (as sheep anti-mouse glomerular basement membrane globulin) in their glomeruli. Crescentic GN did not develop in CD40(-/-) mice due to significantly reduced nephritogenic Th1 responses. IL-12 was administered to CD40(-/-) mice with GN to dissect interactions between IL-12 and CD40 in inducing nephritogenic immunity and injury. Administration of IL-12 to CD40(-/-) mice restored Th cell IFN-gamma production, and up-regulated intrarenal chemokines and glomerular T cell and macrophage accumulation compared with WT control mice. Despite this, renal macrophages were not activated and renal injury and dermal DTH were not restored. Thus, CD40-directed IL-12 drives Th1 generation and effector cell recruitment but CD40 is required for activation. To test this hypothesis, activated OT-II OVA-specific CD4(+) cells and OVA(323-339)-loaded nonresponsive APCs were transferred into footpads of WT, CD40(-/-), and macrophage-depleted WT mice. WT mice developed significant DTH compared with CD40(-/-) and macrophage-depleted WT mice. This study demonstrated that CD40-induced IL-12 is required for generation of systemic Th1 immunity to nephritogenic Ags, and that IL-12 enhances Th1 effector cell recruitment to peripheral sites of Ag presentation via generation of local chemokines. Effector cell activation, renal DTH-like injury, and dermal DTH require direct Th1 CD154/macrophage CD40 interactions.     

IL-18 is redundant in T-cell responses and in joint inflammation in antigen-induced arthritis

IL-18 is an important cofactor in Th1 immune responses and it has additional roles in inflammation. Recent reports suggest the contribution of IL-18 to immune responses may vary between mouse strains and immune contexts. We investigated the contribution of IL-18 to T-cell activation and joint inflammation in Ag-induced arthritis (AIA) in C57Bl/6 mice. AIA and cutaneous delayed-type hypersensitivity (DTH) reactions were induced in wild-type (WT) and IL-18-/- C57Bl/6 mice, and Ag-specific T-cell proliferation and IFN-gamma and IL-4 production were measured. The humoral immune response was measured as serum antibody to the disease-initiating Ag, methylated BSA (mBSA). Splenocyte production of IL-6 was measured by ELISA. To confirm the dependence of this model on Th1-cell-mediated immunity, IL-12p40-/- mice were similarly studied. WT mice developed synovitis, joint effusion, cartilage destruction and bone damage associated with induction of DTH, and in vitro Ag-specific T-cell proliferation and IFN-gamma production. Unexpectedly, IL-18-/- mice developed AIA and indices of T-cell activation were similar to those of WT mice. In contrast, IL-12p40-/- mice did not develop AIA, DTH or T-cell activation. WT and IL-18-/- mice, but not IL-12p40-/- mice, developed significantly increased serum antibody to mBSA compared with naive controls. WT and IL-18-/- splenocytes produced high levels of IL-6, whereas IL-12p40-/- cells had significantly lower IL-6 production compared with both. In conclusion, IL-18 is redundant both as a Th1 response cofactor and inflammatory cytokine, whereas IL-12p40-/- is a key cytokine, in AIA in C57Bl/6 mice.     

IL-12-independent IFN-gamma production by T cells in experimental Chagas' disease is mediated by IL-18.

IL-12p35-deficient (IL-12p35(-/-)) mice were highly susceptible to Trypanosoma cruzi infection and succumbed during acute infection, demonstrating the crucial importance of endogenous IL-12 in resistance to experimental Chagas' disease. Delayed immune responses were observed in mutant mice, although comparable IFN-gamma and TNF-alpha blood levels as in wild-type mice were detected 2 wk postinfection. In vivo and in vitro analysis demonstrated that T cells, but not NK cells, were recruited to infected organs. Analysis of mice double deficient in the recombinase-activating gene 2 (RAG2) and IL-12p35, as well as studies involving T cell depletion, identified CD4(+) T cells as the cellular source for IL-12-independent IFN-gamma production. IL-18 was induced in IL-12p35(-/-) mice and was responsible for IFN-gamma production, as demonstrated by in vivo IL-18 neutralization studies. In conclusion, evidence is presented for an IL-12-independent IFN-gamma production in experimental Chagas' disease that is T cell and IL-18 dependent.     

IL-18-induced expression of intercellular adhesion molecule-1 in human monocytes: involvement in IL-12 and IFN-gamma production in PBMC

IL-18 time- and concentration-dependently upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) in a monocyte population in human PBMC as determined by FACS analysis while the expression of CD11a, CD18, CD29, CD44, and CD62L in monocytes and that of ICAM-1, CD11a, CD18, CD29, CD44, and CD62L in T cells was not influenced by IL-18. IL-18 in the same concentration range stimulated the production of IL-12, TNF-alpha, and IFN-gamma in culture of PBMC; however, IL-18-induced expression of ICAM-1 in monocytes was not inhibited by anti-IL-12, anti-TNF-alpha, or anti-IFN-gamma Ab, suggesting the independence of the upregulating effect of IL-18 on endogenous IL-12, TNF-alpha, and IFN-gamma production. IL-18 also induced the aggregation of PBMC, which was prevented by anti-ICAM-1 and anti-LFA-1 Abs. On the other hand, anti-ICAM-1 and anti-LFA-1 Abs inhibited IL-18-induced production of three cytokines, IL-12, IFN-gamma, and TNF-alpha, by 60 and 40%, respectively. These results strongly suggested that the IL-18-induced upregulation of ICAM-1 and the subsequent adhesive interaction through ICAM-1 on monocytes and LFA-1 on T/NK cells generate an additional stimulatory signaling as well as an efficient paracrine environment for the IL-18-initiated cytokine cascade.     

Prevention of allergen-specific, Th2-biased immune responses in vivo: role of increased IL-12 and IL-18 responsiveness

The factors that control development of adaptive responses to exogenous Ag remain incompletely understood. An ability to selectively direct immunity toward a specific phenotype would be of clinical benefit in numerous immunological disorders. Administration of chemically modified allergen glutaraldehyde-polymerized OVA (OA-POL) leads to >90% reductions in murine IgE and >500-fold increases in IgG2c responses that develop upon subsequent immunization with native Ag. In the present study, we examine the mechanisms underlying this reorientation of the type 2 dominant response that would normally develop. Lack of endogenous IL-12 or IFN-gamma results in markedly reduced induction of IgG2c responses following OA-POL treatment, but only IFN-gamma(-/-) mice demonstrate reduced capacity to prevent IgE induction. This indicates that while both IL-12 and IFN-gamma are critical promoters of type 1 immunity, only IFN-gamma is required to maximally inhibit development of type 2 immune responses. Compared with OVA-immunized mice, CD69(+) T cells from OA-POL-immunized mice demonstrate elevated IL-12Rbeta(2), IL-18Ralpha, and IL-18Rbeta mRNA levels, as well as increased IFN-gamma production in response to rIL-12 or rIL-18 stimulation. Collectively, these data indicate that preventing induction of type 2 immune responses is critically dependent on altered T cell responsiveness to these cytokines. The finding that targeted, Ag-specific manipulation of IL-12 and IL-18 responsiveness can be used to shape the phenotype of the dominant immune response that develops suggests that specifically targeting IL-12 and IL-18 receptor expression may offer clinical options for clinical prophylaxis or intervention.     

Increased susceptibility to immunologically mediated glomerulonephritis in IFN-gamma-deficient mice.

It is postulated that IFN-gamma confers susceptibility to immunologically mediated tissue injury. To test this hypothesis, we compared the intensity of accelerated anti-glomerular basement membrane glomerulonephritis between wild-type (IFN-gamma+/+) and IFN-gamma gene knockout (IFN-gamma-/-) mice. This disease model is initiated by binding of heterologous (sheep) anti-glomerular basement membrane Abs to the glomeruli of mice preimmunized with sheep IgG. The secondary cellular and humoral immune responses to the planted Ag then lead to albuminuria and glomerular pathology. We found that IFN-gamma-/- mice or IFN-gamma+/+ mice injected with IFN-gamma-neutralizing Ab develop worse albuminuria and glomerular pathology than IFN-gamma+/+ mice. The humoral response to sheep IgG (serum mouse anti-sheep IgG titers and intraglomerular mouse IgG deposits) was comparable in the IFN-gamma+/+ and IFN-gamma-/- groups. In contrast, IFN-gamma-/- mice mounted a stronger cellular immune response (cutaneous delayed-type hypersensitivity reaction) to sheep IgG than IFN-gamma+/+ mice. These findings provide evidence that endogenous IFN-gamma has a protective role in immunologically mediated glomerulonephritis initiated by foreign Ags.     

Protective and memory immunity to Histoplasma capsulatum in the absence of IL-10

We determined whether the absence of IL-10 in mice influenced protective and memory immunity to Histoplasma capsulatum. IL-10(-/-) mice cleared primary and secondary infection more rapidly than wild-type controls. Administration of mAb to TNF-alpha or IFN-gamma, but not GM-CSF, abrogated protection in naive IL-10(-/-) mice; mAb to TNF-alpha, but not IFN-gamma or GM-CSF, subverted protective immunity in secondary histoplasmosis. The inflammatory cell composition in IL-10(-/-) mice was altered in those given mAb to IFN-gamma or TNF-alpha. More Gr-1(+) and Mac-3(+) cells were present in lungs of IL-10(-/-) mice given mAb to IFN-gamma, and treatment with mAb to TNF-alpha sharply reduced the number of CD8(+) cells in lungs of IL-10(-/-) mice. We ascertained whether the lack of IL-10 modulated memory T cell generation or the protective function of cells. The percentage of CD3(+), CD44(high), CD62(low), and IFN-gamma(+) cells in IL-10(-/-) mice was higher than that of wild-type at day 7 but not day 21 or 49 after immunization. Fewer splenocytes from immunized IL-10(-/-) mice were required to mediate protection upon adoptive transfer into infected TCR alphabeta(-/-) mice. Hence, deficiency of IL-10 confers a salutary effect on the course of histoplasmosis, and the beneficial effects of IL-10 deficiency require endogenous TNF-alpha and/or IFN-gamma. Memory cell generation was transiently increased in IL-10(-/-) mice, but the protective function conferred by cells from these mice following immunization is strikingly more vigorous than that of wild-type.     

Altered immune responses and susceptibility to Leishmania major and Staphylococcus aureus infection in IL-18-deficient mice.

IL-18, formerly designated IFN-inducing factor, is a novel cytokine produced by activated macrophages. It synergizes with IL-12 in the induction of the development of Th1 cells and NK cells. To define the biological role of IL-18 in vivo, we have constructed a strain of mice lacking IL-18. Homozygous IL-18 knockout (-/-) mice are viable, fertile, and without evident histopathologic abnormalities. However, in contrast to the heterozygous (+/-) or wild-type (+/+) mice, which are highly resistant to the infection of the protozoan parasite Leishmania major, the IL-18-/- mice are uniformly susceptible. The infected IL-18-/- mice produced significantly lower levels of IFN-gamma and larger amounts of IL-4 compared with similarly infected +/- and +/+ mice. In contrast, when infected with the extracellular Gram-positive bacteria Staphylococcus aureus, the IL-18-/- mice developed markedly less septicemia than similarly infected wild-type (+/+) mice. However, the mutant mice developed significantly more severe septic arthritis than the control wild-type mice. This was accompanied by a reduction in the levels of Ag-induced splenic T cell proliferation, decreased IFN-gamma and TNF-alpha synthesis, but increased IL-4 production by the mutant mice compared with the wild-type mice. These results therefore provide direct evidence that IL-18 is not only essential for the host defense against intracellular infection, but it also plays a critical role in regulating the synthesis of inflammatory cytokines, and therefore could be an important target for therapeutic intervention.     

Membranous glomerulonephritis development with Th2-type immune deviations in MRL/lpr mice deficient for IL-27 receptor (WSX-1)

MRL/lpr mice develop spontaneous glomerulonephritis that is essentially identical with diffuse proliferative glomerulonephritis (World Health Organization class IV) in human lupus nephritis. Lupus nephritis is one of the most serious complications of systemic lupus erythematosus. Diffuse proliferative glomerulonephritis is associated with autoimmune responses dominated by Th1 cells producing high levels of IFN-gamma. The initial mounting of Th1 responses depends on the function of the WSX-1 gene, which encodes a subunit of the IL-27R with homology to IL-12R. In mice deficient for the WSX-1 gene, proper Th1 differentiation was impaired and abnormal Th2 skewing was observed during infection with some intracellular pathogens. Disruption of the WSX-1 gene dramatically changed the pathophysiology of glomerulonephritis developing in MRL/lpr mice. WSX-1-/- MRL/lpr mice developed disease resembling human membranous glomerulonephritis (World Health Organization class V) with a predominance of IgG1 in glomerular deposits, accompanied by increased IgG1 and IgE in the sera. T cells in WSX-1-/- MRL/lpr mice displayed significantly reduced IFN-gamma production along with elevated IL-4 expression. Loss of WSX-1 thus favors Th2-type autoimmune responses, suggesting that the Th1/Th2 balance may be a pivotal determinant of human lupus nephritis development.     

IL-12-independent Th1-type immune responses to respiratory viral infection: requirement of IL-18 for IFN-gamma release in the lung but not for the differentiation of viral-reactive Th1-type lymphocytes

We demonstrated that IL-12 was induced during primary or secondary pulmonary adenoviral infection in wild-type (wt) mice. However, cellular responses were not compromised in the lungs of IL-12-/- mice. The level of IFN-gamma in the lung was similar in wt and IL-12-/- mice during pulmonary viral infection. Upon Ag stimulation in vitro, lymphocytes from draining lymph nodes or spleen of infected IL-12-/- mice released large amounts of IFN-gamma, but not IL-4, which were comparable to those released by wt lymphocytes. Furthermore, a predominantly IgG2a response to adenoviral infection was unimpaired in IL-12-/- mice. These significant anti-adenoviral Th1-type responses in IL-12-/- mice led to an efficient clearance of virus-infected cells in the lung. Whether IL-18 was involved in IL-12-independent anti-adenoviral immune responses was investigated. Abrogation of endogenous IL-18 by an Ab resulted in diminished IFN-gamma release and lymphocytic infiltrate in the lung during adenoviral infection. Nevertheless, the development of lymphocytes of the Th1 phenotype was unimpaired in the absence of both IL-12 and IL-18. In contrast to their intact ability to mount Th1-type responses to viral infection, IL-12-/- mice suffered impaired Th1-type immune responses to pulmonary mycobacterial infection. Our findings suggest that IL-12, although induced, is not required for Th1-type responses to respiratory viral infection, in contrast to mycobacterial infection. IL-18 is required for the optimal release of IFN-gamma in the lung during viral infection, but is not required for the generation of virus-reactive Th1-type lymphocytes. Th1 differentiation during respiratory adenoviral infection may involve molecules different from IL-12 or IL-18.     

Interferon-gamma production and host protective response against Mycobacterium tuberculosis in mice lacking both IL-12p40 and IL-18

Interferon (IFN)-gamma plays an essential role in host defense against infection with Mycobacterium tuberculosis, and its synthesis is critically regulated by interleukin (IL)-12, IL-18 and the recently identified IL-23. The present study was designed to determine the roles of these cytokines in IFN-gamma-mediated host defenses against M. tuberculosis. For this purpose, we compared host protective responses in IL-12p40 and IL-18 double-knockout (DKO) mice (which lacked both IL-12/IL-18 and also IL-23) and IFN-gamma gene-disrupted (GKO) mice. DKO mice were more resistant to the infection than GKO mice, as indicated by their extended survival and reduced live colony numbers in spleen, liver and lung. IFN-gamma was detected by ELISA in liver and lung homogenates, but not in spleen and serum, and in all organs by RT-PCR in DKO mice at comparable or reduced levels to those in wild-type mice. IFN-gamma production was reduced by depletion of CD4+ T cells, but not of natural killer (NK), NKT, gammadeltaT and dendritic cells. Neutralization of IFN-gamma or TNF-alpha by specific monoclonal antibodies (mAbs) significantly shortened the survival time of the infected DKO mice. Furthermore, anti-TNF-alpha mAb partially attenuated IFN-gamma synthesis in the liver of these mice. Finally, the expression level of inducible nitric oxide synthase (iNOS) mRNA in the spleen, liver and lung was considerable in DKO mice but only marginal or undetected in GKO mice. Our results indicate the presence of IL-12-, IL-18- and IL-23-independent host protective responses against mycobacterial infection mediated by IFN-gamma, which was secreted from helper T cells.     

IL-18 contributes to host resistance against infection with Pseudomonas aeruginosa through induction of IFN-gamma production

Pseudomonas aeruginosa keratitis destroys the cornea in susceptible (B6), but not resistant (BALB/c) mice. To determine mechanisms mediating resistance, the role of IFN-gamma, IL-12, and IL-18 was tested in BALB/c mice. RT-PCR analysis detected IFN-gamma mRNA expression levels in cornea that were significantly increased at 1-7 days postinfection. IL-18 mRNA was detected constitutively in cornea and, at 1-7 days postinfection, levels were elevated significantly, while no IL-12 mRNA was similarly detected. To test whether IL-18 contributed to IFN-gamma production, mice were treated with anti-IL-18 mAb. Treatment decreased corneal IFN-gamma mRNA levels, and bacterial load and disease increased/worsened, compared with IgG-treated mice. To stringently examine the role of IFN-gamma in bacterial killing, knockout (-/-) vs wild-type (wt) mice also were tested. All corneas perforated, and bacterial load was increased significantly in -/- vs wt mice. Because disease severity was increased in IFN-gamma(-/-) vs IL-18-neutralized mice, and since IL-18 also induces production of TNF, we tested for TNF-alpha in both groups. ELISA analysis demonstrated significantly elevated corneal TNF-alpha protein levels in IFN-gamma(-/-) vs wt mice after infection. In contrast, RT-PCR analysis of IL-18-neutralized vs IgG-treated infected mice revealed decreased corneal TNF-alpha mRNA expression. Next, to resolve whether TNF was required for bacterial killing, TNF-alpha was neutralized in BALB/c mice. No difference in corneal bacterial load was detected in neutralized vs IgG-treated mice. These data provide evidence that IL-18 contributes to the resistance response by induction of IFN-gamma and that IFN-gamma is required for bacterial killing.     

IL-12 alone and in synergy with IL-18 inhibits osteoclast formation in vitro

IL-12, like IL-18, was shown to potently inhibit osteoclast formation in cultures of cocultures of murine osteoblast and spleen cells, as well as in adult spleen cells treated with M-CSF and receptor activator of NF-kappaB ligand (RANKL). Neither IL-12 nor IL-18 was able to inhibit RANKL-induced osteoclast formation in cultured RAW264.7 cells, demonstrating that IL-12, like IL-18, was unable to act directly on osteoclastic precursors. IL-12, like IL-18, was found to act by T cells, since depletion of T cells from the adult spleen cell cultures ablated the inhibitory action of IL-12 and addition of either CD4 or CD8 T cells from C57BL/6 mice to RANKL-stimulated RAW264.7 cultures permitted IL-12 or IL-18 to be inhibitory. Additionally, IL-12 was still able to inhibit osteoclast formation in cocultures with osteoblasts and spleen cells from either GM-CSF R(-/-) mice or IFN-gamma R(-/-) mice, indicating that neither GM-CSF nor IFN-gamma was mediating osteoclast inhibition in these cultures. Combined, IL-18 and IL-12 synergistically inhibited osteoclast formation at concentrations 20- to 1000-fold less, respectively, than when added individually. A candidate inhibitor could not be demonstrated using neutralizing Abs to IL-4, IL-10, or IL-13 or from mRNA expression profiles among known cytokine inhibitors of osteoclastogenesis in response to IL-12 and IL-18 treatment, although the unknown inhibitory molecule was determined to be secreted from T cells.     

Synergistic activation of human monocytes by granulocyte-macrophage colony-stimulating factor and IFN-gamma. Increased TNF-alpha but not IL-1 activity

TNF-alpha and IL-1 activities and PGE2 levels were investigated in the supernatants of highly purified human monocytes cultured for 18 h with recombinant human granulocyte-macrophage CSF (GM-CSF). GM-CSF alone did not stimulate IL-1 or TNF-alpha activities or the production of PGE2. GM-CSF with IFN-gamma, but not with LPS, consistently activated the monocytes for TNF-alpha activity. In contrast, for increased IL-1 activity, GM-CSF synergized weakly and irregularly with LPS, but not at all with IFN-gamma. For the third monocyte product investigated, GM-CSF was a weak and inconsistent inducer of PGE2 and only in the co-presence of IFN-gamma. Thus, GM-CSF can elicit different responses in human monocytes depending both on the co-stimulus as well as the monocyte product being investigated.     

Effects of IL-10 and age on IL-6, IL-1beta, and TNF-alpha responses in mouse skeletal and cardiac muscle to an acute inflammatory insult.

Exaggerated proinflammatory cytokine responses can be observed with aging, and reduced levels of the anti-inflammatory cytokine IL-10 may contribute to these responses. IL-10 can reduce IL-6, IL-1beta, and TNF-alpha expression in nonmuscle tissues; however, no studies have examined the combined effects of IL-10 and age on cytokine responses in skeletal and cardiac muscle. These experiments tested the hypothesis that the absence of IL-10, in vivo, is associated with greater IL-6, TNF-alpha, and IL-1beta responses to an inflammatory challenge in skeletal and cardiac muscle and that aging exaggerates these responses. We compared IL-6, IL-1beta, and TNF-alpha mRNA and protein levels in skeletal and cardiac muscle of young (4 mo) and mature (10-11 mo) wild-type (IL-10(+/+)) and IL-10 deficient (IL-10(-/-)) mice following LPS. Skeletal and cardiac IL-6 mRNA and protein were elevated by LPS for IL-10(+/+) and IL-10(-/-) mice with greater responses in the IL-10(-/-) mice (P < 0.01). In skeletal muscle these effects were greater in mature than young mice (P < 0.01). IL-1beta mRNA and protein responses to LPS were greater in cardiac muscle of young but not mature IL-10(-/-) mice compared with IL-10(+/+) (P < 0.01). However, IL-1beta responses were greater in mature than young mice, but only in IL-10(+/+) groups (P < 0.05). The absence of IL-10 was associated with higher TNF-alpha protein levels in cardiac muscle (P < 0.05). The results provide the first in vivo evidence that the absence of IL-10 is associated with a greater IL-6 response to LPS in skeletal and cardiac muscles, and in skeletal muscle aging further exaggerates these responses.     

Proinflammatory properties of IL-4 in the intestinal microenvironment

IL-4 is involved in type 2 T helper cell (Th)2-type immune responses and, in some cases, can promote Th1 responses. However, the proinflammatory potential of IL-4 alone is unclear. In this study, we examined the ability of IL-4 to induce colitis after its overexpression in the colon using an adenoviral vector (Ad5) and compared results with those obtained after overexpression of IL-12, a cytokine implicated in several models of colitis. Overexpression of IL-4 or IL-12 caused a fatal colitis within 24 h in 60% of animals and was dose and strain dependent. IL-12-induced colitis was accompanied by the local expression of IFN-gamma and TNF-alpha but not IL-4 mRNA and protein. Conversely, IL-4-induced colitis was accompanied by the local expression of IL-4 and TNF-alpha but not IFN-gamma mRNA and protein. The Ad5-IL4-induced colitis did not persist beyond 3 days and was present in recombinase activation gene-2 (RAG-2)-/- mice but not in STAT6-/- mice. Acute lethal colitis induced by Ad5IL12 was T cell mediated and IFN-gamma receptor (IFN-gamma R) dependent. Furthermore, TNF-alpha was found to be important in the pathogenesis of Ad5IL-4 and Ad5IL-12-induced colitis. Results of this study indicate that IL-4 alone can act as a proinflammatory cytokine in the gut of normal mice, inducing a rapid onset and short-lived colonic injury while maintaining a Th2-type cytokine profile that functions via a local T cell-independent mechanism involving TNF-alpha.     

Combined effects of IL-12 and IL-18 on the induction of collagen-induced arthritis

IL-18 expression has recently been detected in rheumatoid arthritis (RA) synovial membrane. We investigated the mechanisms by which IL-18-induced collagen-induced arthritis in DBA/1 mice primed intradermally with type II bovine collagen in IFA and boosted i.p. 21 days later with CII in saline. Mice were injected i.p. with rIL-12, rIL-18, or both (100 ng) during days -1 to 4 and again on days 20-24. Control mice received PBS. Mice treated with IL-12 or IL-18 alone developed significantly higher incidence and more severe disease compared with controls. These were elevated further by combination treatment with IL-12 and IL-18. The cytokine treatments led to markedly enhanced synovial hyperplasia, cellular infiltration, and cartilage erosion compared with controls. Cytokine-treated mice produced significantly more IFN-gamma, TNF-alpha, and IL-6 than the controls. Interestingly, IL-18-treated mice produced more TNF-alpha and IL-6, but less IFN-gamma, compared with mice treated with IL-12. Furthermore, splenic macrophages from DBA/1 mice cultured in vitro with IL-18, but not IL-12, produced substantial amounts of TNF-alpha. Mice treated with IL-18 or IL-18 plus IL-12 produced markedly more IgG1 and IgG2a anti-collagen Ab compared with controls, whereas IL-12 treatment only led to an enhanced IgG2a response. Together these results demonstrate that IL-18 can promote collagen-induced inflammatory arthritis through mechanisms that may be distinct from those induced by IL-12.     

IL-4 and IL-10 are essential for immunosuppression induced by high molecular weight proteins from Ascaris suum

The extract from Ascaris suum worms (Asc) impairs Th1 and Th2 responses to a non-related antigen, i.e. ovalbumin (OVA). Its suppressive capacity is due to high molecular weight components present in a gel filtration fraction (PI). This fraction is able to elicit IL-4 and IL-10 secretion. Interestingly enough, it induces anti-PI non-anaphylactic IgG1 synthesis through the action of IL-12/IFN-gamma. Here, we investigated the down-regulation of the immune response to OVA by PI in IL-12, IFN-gamma, IL-4 or IL-10 C57BL/6 knockout mice immunized with OVA+PI in adjuvant. OVA-induced delayed-type hypersensitivity (DTH) reactions, secretion of IL-2 and IFN-gamma, and IgG1, IgG2c and IgE antibody production were suppressed by PI in wild-type mice, as well as in IL-12- or IFN-gamma-deficient mice. In contrast, PI had no effect on anti-OVA IgE production and DTH, and induced only a partial suppression of IgG1 and IFN-gamma in IL-10(-/-) mice. The experiments also showed that IL-4 was involved in the PI-induced suppression of IgG2c antibodies and IL-2 secretion. Finally, down-regulation of IFN-gamma was not seen in mice lacking both IL-4 and IL-10, i.e. IL-4(-/-) mice treated with anti-IL-10 antibodies before immunization. These results exclude the participation of IL-12 and IFN-gamma in PI-induced immunosuppression, and highlight the essential role of IL-10 in the suppression of OVA-specific Th2-related parameters, as well as the cooperation between IL-10 and IL-4 in the suppression of Th1-related parameters.