IL-15 promotes IL-12 production by human monocytes via T cell-dependent contact and may contribute to IL-12-mediated IFN-gamma secretion by CD4+ T cells in the absence of TCR ligation

At inflammatory sites, the number of activated bystander T cells exceeds that of Ag-activated T cells. We investigated whether IL-15, a monocyte-derived cytokine that shares several biologic activities with IL-2, may contribute to bystander T cell activation in the absence of IL-2 and triggering Ag. The addition of IL-15 to cocultures of monocytes and T cells stimulates CD4+ but not CD8+ T cells to produce IFN-gamma. IFN-gamma production requires endogenous IL-12, the production of which in turn is dependent upon CD40/CD154 interactions between CD4+ T cells and monocytes. Indeed, non-TCR-activated CD4+ but not CD8+ T cells express significant levels of CD154. IL-15 may enhance IFN-gamma in this system by up-regulating CD40 expression on monocytes and IL-12Rbeta1 expression on CD4+ T cells. Conversely, using neutralizing anti-IL-15 mAb, we show that the ability of IL-12 to augment IFN-gamma secretion is partly mediated by endogenous IL-15. Finally, in the absence of monocytes, a synergistic effect between exogenous IL-12 and IL-15 is necessary to induce IFN-gamma production by purified CD4+ T cells, while IL-15 alone induces T cell proliferation. It is proposed that this codependence between IL-12 and IL-15 for the activation of inflammatory T cells may be involved in chronic inflammatory disorders that are dominated by a Th1 response. In such a response, a self-perpetuating cycle of inflammation is set forth, because IL-15-stimulated CD4+ T cells may activate monocytes to release IL-12 that synergizes with IL-15 to induce IL-12 response and IFN-gamma production.     

Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection

The intracellular protozoan Toxoplasma gondii is a widespread opportunistic parasite of humans and animals. Normally, T. gondii establishes itself within brain and skeletal muscle tissues, persisting for the life of the host. Initiating and sustaining strong T-cell-mediated immunity is crucial in preventing the emergence of T. gondii as a serious pathogen. The parasite induces high levels of gamma interferon (IFN-gamma) during initial infection as a result of early T-cell as well as natural killer (NK) cell activation. Induction of interleukin-12 by macrophages is a major mechanism driving early IFN-gamma synthesis. The latter cytokine, in addition to promoting the differentiation of Th1 effectors, is important in macrophage activation and acquisition of microbicidal functions, such as nitric oxide release. During chronic infection, parasite-specific T lymphocytes release high levels of IFN-gamma, which is required to prevent cyst reactivation. T-cell-mediated cytolytic activity against infected cells, while easily demonstrable, plays a secondary role to inflammatory cytokine production. While part of the clinical manifestations of toxoplasmosis results from direct tissue destruction by the parasite, inflammatory cytokine-mediated immunopathologic changes may also contribute to disease progression.     

The role of T cells in polyethylene particulate induced inflammation

OBJECTIVE: To investigate the role of T lymphocytes in ultra-high molecular weight polyethylene (UHMWPE) induced inflammation in joint arthroplasty. METHOD: We address the role of T cells in wear induced inflammation by injecting the knee joints of both immune competent rats and mice and severe combined immunodeficient (SCID) mice with UHMWPE. Histological and immunohistochemical analysis of the synovial tissues was compared. Interaction between human T cells and UHMWPE particles was examined in vitro using T cell activation assays. RESULTS: Histological and immunohistochemical analysis of the knees of the immune competent animals showed significant UHMWPE induced inflammation. In contrast, the tissue in the SCID mice knee joints showed very little inflammatory response to UHMWPE despite phagocytosis of the particulate. Since the SCID mice have no functional T or B lymphocytes, it is highly likely that the lack of inflammation in knee joints may be due to the absence of mouse T cells, as the infiltration of T cells into the joint tissue may enhance the inflammatory response to UHMWPE particles. T cell activation assays showed that T cells were not directly activated by UHMWPE particles and the nature of the interaction was not revealed from these experiments. CONCLUSIONS: Although T cells are not directly involved in UHMWPE particle induced inflammation, as shown by the T cell activation assays, the histological data from the mice studies clearly show differences in the amplitude of inflammation from animals with and without functional T cells. Our studies suggest that the T cells may enhance the inflammatory response due to a bystander effect. Since the macrophages upon ingestion of UHMWPE particles release several cytokines including tumor necrosis factor-alpha, interleukin 1, and IL-6, it is possible that T cells in the vicinity of these macrophages may become attracted to the knee joint and activated due to cytokine release.     

IL-12 is not required for induction of type 1 cytokine responses in viral infections

To investigate the physiological role of IL-12 in viral infections in terms of T cell cytokine responses involved in virus-specific Ig isotype induction and in antiviral protection, immune responses elicited upon infection of IL-12-deficient mice with lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus (VSV) were studied. Infection of IL-12-deficient mice with LCMV induced a virus-specific type 1 cytokine response as determined by in vitro cytokine secretion patterns as well as by in vivo intracellular cytokine staining of LCMV-specific CD4+ TCR transgenic T cells that had clonally expanded in LCMV-infected IL-12-deficient recipient mice. In addition, LCMV- and VSV-specific IgG responses exhibited normal serum IgG2a/IgG1 ratios, demonstrating again virus-specific CD4+ T cell induction of type 1 phenotype in IL-12-deficient mice upon viral infection. LCMV and VSV immune mice were found to be protected against challenge immunization with recombinant vaccinia viruses expressing either the LCMV- or the VSV-derived glycoprotein, respectively. This protection is known to be mediated by T cell-secreted type 1 cytokines IFN-gamma and TNF-alpha. In contrast, IL-12-deficient mice showed impaired abilities to control infection with the facultative intracellular bacterium Listeria monocytogenes at early time points after infection. However, at later time points of infection, IL-12-deficient mice were able to clear infection. These findings may indicate that viruses are able to induce type 1 T cell responses in the absence of IL-12 as opposed to some bacterial or parasitical infections that are crucially dependent on the presence of IL-12 for the induction of type 1 immune responses.     

Blockade of CTLA-4 enhances host resistance to the intracellular pathogen, Leishmania donovani

CTLA-4 has recently been shown to act as a negative regulator of T cell activation. Here we provide evidence that blockade of CTLA-4 can result in enhanced host resistance to an intracellular pathogen. The administration of anti-CTLA-4 mAb 4F10 to BALB/c mice, 1 day following infection with Leishmania donovani, enhanced the frequency of IFN-gamma and IL-4 producing cells in both spleen and liver, and dramatically accelerated the development of a hepatic granulomatous response. The expression of mRNA for the CXC chemokine gammaIP-10 was also elevated above that seen in control Ab treated mice, and was directly correlated with the frequency of IFN-gamma producing cells. In contrast, macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1) mRNA levels were unaffected by anti-CTLA-4 treatment, suggesting that CTLA-4 blockade may exert selective effects on chemokine expression. These changes in tissue response and cytokine/chemokine production were accompanied by a 50 to 75% reduction of parasite load in the spleen and liver of anti-CTLA-4-treated animals compared to controls. Furthermore, administration of anti-CTLA-4 mAb 15 days after L. donovani infection, when parasite burden is increasing in both organs, also resulted in enhanced resistance. Thus, these studies indicate a potent immunomodulatory and potentially therapeutic role for interventions targeted at CTLA-4.     

The mRNA-phenotype of granuloma formation: CD4+ T cell-associated cytokine gene expression during primary murine listeriosis

In murine listeriosis, elimination of bacteria and immunity to reinfection critically depend on Thy1+ CD4- cells, while cell-mediated inflammatory phenomena such as DTH and granuloma formation are mostly mediated by CD4+ T cells. In an attempt to correlate T cell phenotype and function with a particular set of cytokines produced, we examined the cytokine gene expression profile associated with the presence or absence of Thy1+, CD4+ and/or CD8+ cells in the livers of mice during a primary infection with L. monocytogenes. The presence of CD4+ cells was found to be closely associated with mRNA expression for IL-2, IL-3 and IL-4, a 5-fold increase in expression of TNF-alpha and GM-CSF and a 25-fold increase in expression of IFN-gamma and TNF-beta mRNAs, and temporally coincided with the development of granulomatous lesions. In vivo neutralization of TNF-alpha and, to a lesser extent, IFN-gamma resulted in abrogation of granuloma formation. A similar correlation between the presence of CD8+ cells and mRNA expression for any one of the cytokines studied did not exist, pointing to a qualitatively different mechanism of CD8+ T cell mediated cure of listeriosis.     

Regulation of mouse bone marrow macrophage mannose receptor expression and activation by prostaglandin E and IFN-gamma

The macrophage mannose receptor mediates the clearance of microorganisms and glycoproteins containing terminal mannose oligosaccharides. Cell surface expression of this receptor progresses with macrophage differentiation, and thus may be critical to the scavenger function of tissue and circulating macrophages. Bone marrow macrophages, which were used in this study, differentiate in culture and express functional mannose receptors. The cytokine IFN-gamma triggered activation of these macrophages and down-regulated cell surface expression of the mannose receptor after 48 h. Macrophage activation, as assessed by the generation of superoxide radicals, was inversely correlated with mannose receptor expression. The number of surface receptors was diminished by exposure to IFN-gamma, whereas the binding affinity of the mannose receptor remained unchanged. Treatment with IFN-gamma reduced receptor biosynthesis yet did not alter receptor degradation. Mannose receptor biosynthesis is up-regulated by PG of the E series, and these anti-inflammatory agents reversed the effects of IFN-gamma on receptor expression. Down-regulation of the mannose receptor by IFN-gamma was fully reversible by PGE, indicating that receptor levels are dependent on the functional state of the cell rather than being linked to terminal cell differentiation. The regulation of the receptor by cytokines and anti-inflammatory reagents suggests that the mannose receptor plays a critical role in macrophage scavenger functions and potentially in modulating inflammatory reactions.     

Features of Schistosoma mansoni infection in SCID mice

Features of Schistosoma mansoni infection in SCID mice, which lack functional T- and B-lymphocytes, were investigated. The retarded development of parasites as well as reduction of liver egg recovery in SCID mice was significantly lower than those in congenic counterpart C.B-17 mice. Furthermore, the rate of parasite recovery from SCID mice with primary infection was always lower than that from C.B-17 mice by 20%, showing the innate resistance to S. mansoni infection. SCID mice vaccinated with UV-attenuated S. mansoni cercariae did not show protective immunity against a homologous challenge infection. The present innate resistance exhibited in SCID mice is discussed in relation to cell mediated immunity of macrophage activation by IFN-gamma which would not involve T-lymphocytes but is initiated by IL-12 and TNF-alpha cytokines. SCID mice may provide novel information on the host-parasite relationship in schistosome infections.     

Neutralization of IL-12 decreases resistance to Listeria in SCID and C.B-17 mice. Reversal by IFN-gamma

Interleukin-12 (IL-12) is necessary for the production of IFN-gamma by NK cells during the generation of innate immunity and by T cells for the development of the Th1 response during specific cell-mediated immunity. Here we demonstrate that the endogenous production of IL-12 is critical to the survival of both immunocompromised SCID mice and normal C.B-17 control mice during a primary infection with Listeria monocytogenes. When IL-12 is neutralized in vivo, both strains of mice die at a normally sublethal dose of Listeria. Anti-IL-12 antibody-treated mice showed a decrease in macrophage I-Ad expression and an increase Listeria burden in the spleen. Furthermore, as has been demonstrated in vitro, these effects of IL-12 in vivo were predominantly regulated through the production of IFN-gamma. Administration of IFN-gamma simultaneously with neutralizing antibodies to IL-12 restored macrophage I-Ad expression, limited the spread of the infection, and resulted in the survival of SCID mice. Thus, IL-12 is critical for resistance to infection with Listeria monocytogenes, and this resistance is mediated through stimulation by IL-12 of IFN-gamma production. Concomitant experiments confirmed that anti-TNF antibodies also resulted in uncontrolled infection and a decrease in macrophage I-Ad expression. However, administration of IFN-gamma restored the levels of I-Ad in macrophages but did not limit Listeria growth.     

T cell stimulus-induced crosstalk between lymphocytes and liver macrophages results in augmented cytokine release

Polyclonal T cell stimulation in humans leads to a cytokine burst syndrome that may result in organ failure or lethality. Mechanisms of such cytokine-dependent morbidity can be studied in mice challenged with the T cell mitogen concanavalin A (Con A). In this model tumor necrosis factor (TNF)-dependent toxicity is characterized by a relatively selective liver failure. We examined here whether a crosstalk between liver macrophages and lymphocytes may be the underlying cause for the overshooting TNF response. Lymphocytes from lymph nodes, thymus, or the spleen were cocultured with Kupffer cells and stimulated with the polyclonal T cell stimuli Con A, anti-CD3 mAb, or staphylococcal enterotoxin B. We observed a rapid and synergistically augmented release of TNF, and also of IL-1, IL-2, IL-4, IL-6, and IFN-gamma, compared to stimulation of the individual cell types alone. This dramatically upregulated cytokine response did not require direct cell contact, but was mediated by a soluble factor. In order to find out whether TNF upregulation would require additional cell types in the liver, we used cocultures of T cells and a macrophage cell line and confirmed our previous results. In this model system an increase in TNF mRNA was observed in macrophages, but not in T cells. We conclude that the T cell-macrophage crosstalk following polyclonal T cell stimulation may be responsible for an overshooting TNF release from macrophages. This mechanism finally may lead to organ damage such as liver injury upon Con A injection into mice.     

Lack of both types 1 and 2 cytokines, tissue inflammatory responses, and immune protection during pulmonary infection by Mycobacterium bovis bacille Calmette-Guérin in IL-12-deficient mice

Understanding of key cytokines and the nature of protective immune responses in pulmonary mycobacterial diseases remains a task of paramount importance. In this study, both wild-type (wt) and IL-12-deficient (IL-12(-/-)) mice were infected by airways inoculation of live Mycobacterium bovis bacille Calmette-Guérin (BCG). The type 1 cytokines IL-12, IFN-gamma, and TNF-alpha, but not the type 2 cytokines IL-4 and granulocyte macrophage (GM)-CSF, markedly increased in the lung and peripheral blood of wt mice postinfection, which resulted in the development of intense granulomatous responses and the effective control of mycobacterial infection in the lung. In contrast, IL-12(-/-) mice demonstrated a lack of both types 1 and 2 cytokines in the lung and blood and a severely impaired tissue immune-inflammatory response lacking not only macrophages and neutrophils but CD4 and CD8 T cells and NK cells in the lung throughout the entire course of study. Total lung mononuclear cells isolated from these mice, in contrast to wt mice, had an impaired recall immune response to Ag challenge in vitro. These impaired responses resulted in an uncontrolled local growth and systemic spread of bacilli. Our findings reveal that IL-12 plays an irreplaceable role in the initiation of Th1 responses, and the loss of its function cannot be compensated for by alternative mechanisms in the lung. This cytokine, together with IFN-gamma and TNF-alpha, and granulomatous inflammation are critically required for the effective control of pulmonary mycobacterial infection. Our results also indicate that the absence of type 1 cytokines does not necessarily favor a Th2 response.     

IL-10 gene knockout mice show enhanced Th1-like protective immunity and absent granuloma formation following Chlamydia trachomatis lung infection

We previously reported that higher IL-10 production is correlated with lower IFN-gamma production, weaker delayed hypersensitivity (DTH), and slower organism clearance following chlamydial infection in mice. To assess more directly the role of IL-10, we examined protective immunity and pathological reaction in C57BL/6 IL-10 gene knockout (KO) and wild-type mice. The results showed that in the absence of endogenous IL-10, mice had significantly accelerated chlamydial clearance and developed significantly stronger DTH responses, which could be inhibited by local delivery of rIL-10. Consistent with the enhancement of DTH responses, IL-10 KO mice showed stronger and more persistent CD4 T cell-dependent IFN-gamma production and significant elevation of IL-12 and TNF-alpha production. Additionally, wild-type, but not IL-10 KO, mice showed granuloma formation that was correlated with higher levels of Th2 cytokine (IL-5) production at the later stages of infection. Moreover, chlamydial infection, unlike parasitic protozoan infection, did not induce significant acute toxicity in IL-10 KO mice, which may be due to the low (undetectable) levels of systemic release of proinflammatory cytokines. These results suggest that IL-10 inhibits the priming and expansion of Th1-like T cell responses and that IL-10 plays a role in the fibrotic reaction seen with chlamydial infection.     

Evaluation of IL-12 in immunotherapy and vaccine design in experimental Mycobacterium avium infections

IL-12 is a pivotal cytokine in the induction of IFN-gamma-mediated protective immune responses. We tested the effects of rIL-12 administration to Mycobacterium avium-infected mice and found a limited ability to induce protection against the infection; this ability varied according to the mycobacterial strain studied. IL-12 accelerated the expression and production of IFN-gamma in both immunocompetent and immunodeficient SCID or CD4-depleted mice. Evidence of NK cell activation was found as well as an enhancement of the ability to adoptively transfer resistance with T cell-enriched spleen cell populations and an increase in inflammatory cell recruitment in the liver. The protective ability of IL-12 was dependent upon the endogenous production of IFN-gamma as evaluated by the use of specific neutralizing Abs or IFN-gamma gene-disrupted mice. IL-12 potentiated the protective immunity conferred by a subunit vaccine containing M. avium culture filtrate proteins and dimethyl dioctadecyl ammonium chloride as an adjuvant. Thus, we show limited immunotherapeutic benefits from IL-12 administration in M. avium infections and promising results in its use as a coadjuvant in vaccine design.     

Lysophosphatidylcholine upregulates CD40 ligand expression in newly activated human CD4+ T cells

Lysophosphatidylcholine (lyso-PC) accumulates in tissues undergoing inflammation and atherosclerosis, where an infiltration of T cells is also seen. We found that lyso-PC increased IFN-gamma production and CD40L expression in CD4+ T cells stimulated with anti-CD3 Ab and recombinant CD80 molecules, whereas lyso-PC did not affect IL-2 and IL-4 production. These results suggest that lyso-PC, in combination with other stimuli, may regulate CD4+ T cell functions to propagate local inflammatory reactions and also imply a novel role played by a modified lipid in the selection of Th1/Th2 immune response as well as in the T cell mediated pathogenesis in atherosclerosis.     

The inflammatory response to nonfatal Sindbis virus infection of the nervous system is more severe in SJL than in BALB/c mice and is associated with low levels of IL-4 mRNA and high levels of IL-10-producing CD4+ T cells

SJL mice are susceptible to inflammatory autoimmune diseases of the central nervous system (CNS), while BALB/c mice are relatively resistant. To understand differences in immune responses that may contribute to autoimmune neurologic disease, we compared the responses of SJL and BALB/c mice to infection with Sindbis virus, a virus that causes acute nonfatal encephalomyelitis in both strains of mice. Clearance of virus was similar, but SJL mice developed a more intense inflammatory response in the brain and spinal cord and inflammation persisted for several weeks. Analysis of lymphocytes isolated from brains early after infection showed an absence of NK cells in SJL mice, while both strains of mice showed CD4+ and CD8+ T cells. During the second week after infection, CD4+ T cells increased in SJL mice and the proportion of CD8+ T cells decreased, while the opposite pattern was seen in BALB/c mice. Expression of IL-10 mRNA was higher and IL-4 mRNA was lower in the brains of infected SJL than in BALB/c mice, while expression of the mRNAs of IL-6, IL-1beta, TNFalpha, and the Th1 cytokines IL-2, IL-12, and IFN-gamma was similar. Lymphocytes isolated from the CNS of SJL mice produced large amounts of IL-10. CNS lymphocytes from both strains of mice produced IFN-gamma in response to stimulation with Sindbis virus, but not in response to myelin basic protein. These data suggest that IL-10-producing CD4+ T cells are differentially recruited to or regulated within the CNS of SJL mice compared with BALB/c mice infected with Sindbis virus, a characteristic that may be related to low levels of IL-4, and is likely to be involved in susceptibility of SJL mice to CNS inflammatory diseases.     

Interleukin-12 inhibits hepatitis B virus replication in transgenic mice

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells that has the ability to induce gamma interferon (IFN-gamma) secretion by T and natural killer cells and to generate normal Th1 responses. These properties suggest that IL-12 may play an important role in the immune response to many viruses, including hepatitis B virus (HBV). Recently, we have shown that HBV-specific cytotoxic T lymphocytes inhibit HBV replication in the livers of transgenic mice by a noncytolytic process that is mediated in part by IFN-gamma. In the current study, we demonstrated that the same antiviral response can be initiated by recombinant murine IL-12 and we showed that the antiviral effect of IL-12 extends to extrahepatic sites such as the kidney. Southern blot analyses revealed the complete disappearance of HBV replicative intermediates from liver and kidney tissues at IL-12 doses that induce little or no inflammation in these tissues. In addition, immunohistochemical analysis demonstrated the disappearance of cytoplasmic hepatitis B core antigen from both tissues after IL-12 treatment, suggesting that IL-12 either prevents the assembly or triggers the degradation of the nucleocapsid particles within which HBV replication occurs. Importantly, we demonstrated that although IFN-gamma, tumor necrosis factor alpha, and IFN-alpha/beta mRNA are induced in the liver and kidney after IL-12 administration, the antiviral effect of IL-12 is mediated principally by its ability to induce IFN-gamma production in this model. These results suggest that IL-12, through its ability to induce IFN-gamma, probably plays an important role in the antiviral immune response to HBV during natural infection. Further, since relatively nontoxic doses of recombinant IL-12 profoundly inhibit HBV replication in the liver and extrahepatic sites in this model, IL-12 may have therapeutic value as an antiviral agent for the treatment of chronic HBV infection.