Immune complexes are potent inhibitors of interleukin-12 secretion by human monocytes

We have studied the effect of immune complexes (IC) on interleukin (IL)-12 secretion by human monocytes in vitro. Two experimental models of IC were used. IC formed of tetanus toxoid and polyclonal anti-tetanus toxoid antiserum as well as heat-aggregated human serum IgG almost completely inhibited IL-12 (p70 and p40) secretion induced by interferon-γ and lipopolysaccharide in human blood-derived monocytes. Neutralizing anti-IL-10 antibodies plus indomethacin restored IL-12 secretion in the presence of IC to a high extent, indicating that IL-10 and prostaglandin (PG) partially mediate the IC-induced inhibition of IL-12 secretion. However, neutralization of tumor necrosis factor (TNF)-α by specific antibodies also incompletely restored IL-12 secretion. Indeed, monocytes secrete high levels of TNF-α upon stimulation by IC. We found that exogenously added TNF-α caused a profound inhibition of monocytic IL-12 secretion in the absence of IC, again mediated via the induction of IL-10 and PG. In summary, IC inhibit IL-12 secretion via TNF-α-induced IL-10 and PG synthesis. We conclude that IC, typically appearing in the course of chronic inflammatory processes, may influence the balance between Th1 and Th2 responses and may thus contribute to a deprivation of cell-mediated immune responses.     

Effect of interleukin-10 on dendritic cell maturation and function

The main function of dendritic cells (DC) is to induce the differentiation of naive T lymphocytes into helper cells producing a large array of lymphokines, including interleukin (IL)-2; interferon-γ (IFN-γ), IL-4, IL-5 and IL-10. The potent immunostimulatory properties of DC develop during a process of maturation that occurs spontaneously in vitro. Since IL-10 has been shown to inhibit Th1 responses, we determined its effect on DC maturation and accessory function. Our data show that DC that have undergone maturation in vitro in the presence of IL-10, have an impaired capacity to induce a Th1-type response in vivo, leading to the development of Th2 lymphocytes. Their inability to promote the synthesis of IFN-γ seems to correlate with a decreased production of IL-12, an heterodimeric cytokine necessary for optimal generation of Th1-type cells. These results suggest that IL-10 skews the Th1/Th2 balance to Th2 in vivo by selectively blocking IL-12 synthesis by the antigen-presenting cells that play a role of adjuvant of the primary immune response. The cytokines present in the environment at the presentation step may, therefore, determine the class of the immune response induced by DC in vivo, i.e. Th0 Th1 and/or Th2.     

Contribution of interleukin-3 to antigen-induced Th2 cytokine production

Short-term stimulation of mouse spleen cells in vitro with interleukin (IL)-3 induces the secretion of the Th2 cytokines IL-4 and IL-6. Non-B/non-T cells were the target of this IL-3 effect. However, during long-term antigen-dependent culture, T cells are the major source of IL-4 and IL-6. The addition of IL-3 to such cultures also led to a significant increase in IL-4 and IL-6 production. This Th2 cytokine secretion was amplified by the addition of irradiated non-B/non-T cells at the initiation of culture, and was inhibited by anti-IL-4 antibodies. These findings suggest that IL-3 induces the rapid release of IL-4 and IL-6 by non-B/non-T cells, thereby creating an immune milieu conducive to the development of antigen-specific IL-4 and IL-6-secreting Th2 cells.     

Progressive disease or protective immunity to Leishmania major infection: the result of a network of stimulatory and inhibitory interactions

 The study of experimental infection of inbred strains of mice with the intracellular protozoan parasite Leishmania major has contributed significantly not only to our understanding of this fascinating host/parasite relationship but also to that of many basic immunological phenomena. Much has been learned about the cognate interaction of antigen-specific T cells and antigen-presenting cells, about cytokine and T cell subset regulation, and the requirements for costimulation. Specifically, the immune response to experimental L. major infection is the paradigm for polarized T helper cell (Th) 1 and Th2 differentiation. In this model system a Th1 response characterized by interleukin (IL)-2 and interferon (IFN)-γ secretion leads to self-curing disease, whereas a Th2 response (IL-4, IL-10) leads to nonhealing disease. Numerous manipulations, including the injection of cytokines and of neutralizing anti-cytokine antibodies, cytokine transgene expression, and more recently cytokine and cytokine receptor gene knockout studies, have all provided intriguing new pieces to the still incomplete mosaic of our understanding of the immune response. Some of these findings were clearly unexpected and are still incompletely understood. For instance, based on earlier neutralizing anti-IL-4 monoclonal antibody injection studies, IL-4 gene-disrupted BALB/c mice were expected to be unable to mount the biased Th2 response typical of the IL-4^+/+ wild-type mice and to be able to control their lesions; quite unexpectedly, the BALB/c IL-4 knockout mice remain unable to heal their L. major infection. Based on these unexpected findings, we reexamine the literature in an attempt to resolve this apparent paradox and to relate the large body of experimental findings in the mouse system to that which is known about natural and experimental infections in the human. Received: 18 February 1997 / Accepted: 31 July 1997     

Interleukin-4 and interleukin-10 synergize to inhibit cell-mediated immunity in vivo

The lack of cell-mediated (Thl-like) immunity that is often associated with strong humoral immune responses is thought to be due in part to the inhibition of Th1 effector function by the Th2-derived cytokine interleukin-10 (IL-10). This hypothesis, however, is based entirely on results from in vitro studies, wherein IL-10 has been shown to inhibit Thl cytokine synthesis. In this study we have compared the regulatory effects of both IL-4 and IL-10 on the development of a more complex Thl effector function in vivo, the development of delayed-type hypersensitivity (DTH) to Leishmania major in mice immune to Leishmania. The results revealed two findings unexpected from in vitro studies with Thl clones. First, optimal inhibition of the DTH response (up to 70 %), assessed by footpad swelling and leukocytic infiltration, required the combination of IL-4 and IL-10, indicating that these two activities synergized to inhibit DTH reactivity. Second, IL-4 inhibited interferon-γ (IFN-γ) production by lymph node cells draining the site of antigen challenge as well as did IL-10. The combination of both cytokines was no more effective than either alone. The mechanism by which IL-4 and IL-10 acted to inhibit DTH responses did not appear to be through inhibition of IFN-y or tumor necrosis factor production as treatment with antibodies which neutralized these activities failed to inhibit DTH responses. Inhibition of the DTH with IL-4 and IL-10 is the most effective specific regulator of DTH responses reported and the only one capable of modulating tuberculin DTH. These data establish IL-4 and IL-10 as potent inhibitors of Thl effector function in vivo and suggest their utility in controlling deleterious Thl-mediated inflammatory responses such as occur in some infectious and autoimmune diseases.     

Interleukin-4 and interleukin-10 synergize to inhibit cell-mediated immunity in vivo

The lack of cell-mediated (Thl-like) immunity that is often associated with strong humoral immune responses is thought to be due in part to the inhibition of Thl effector function by the Th2-derived cytokine interleukin-10 (IL-10). This hypothesis, however, is based entirely on results from in vitro studies, wherein IL-10 has been shown to inhibit Thl cytokine synthesis. In this study we have compared the regulatory effects of both IL-4 and IL-10 on the development of a more complex Thl effector function in vivo, the development of delayed-type hypersensitivity (DTH) to Leishmania major in mice immune to Leishmania. The results revealed two findings unexpected from in vitro studies with Thl clones. First, optimal inhibition of the DTH response (up to 70 %), assessed by footpad swelling and leukocytic infiltration, required the combination of IL-4 and IL-10, indicating that these two activities synergized to inhibit DTH reactivity. Second, IL-4 inhibited interferon-γ (IFN-γ) production by lymph node cells draining the site of antigen challenge as well as did IL-10. The combination of both cytokines was no more effective than either alone. The mechanism by which IL-4 and IL-10 acted to inhibit DTH responses did not appear to be through inhibition of IFN-γ or tumor necrosis factor production as treatment with antibodies which neutralized these activities failed to inhibit DTH responses. Inhibition of the DTH with IL-4 and IL-10 is the most effective specific regulator of DTH responses reported and the only one capable of modulating tuberculin DTH. These data establish IL-4 and IL-10 as potent inhibitors of Thl effector function in vivo and suggest their utility in controlling deleterious Thl-mediated inflammatory responses such as occur in some infectious and autoimmune diseases.     

A Mixed Th1/Th2 Cell Cytokine Response Predominates in Systemic Onset Juvenile Rheumatoid Arthritis: Immunoregulatory IL-10 Function

The immune response identified by the induction of Th1/Th2 cells plays a critical role in the pathogenesis of various inflammatory and immune disorders. We have determined that in children with systemic onset juvenile rheumatoid arthritis (JRA), peripheral blood mononuclear cells (PBMC) constitutively and after stimulation with various antigensin vitroinduce a higher secretion of interleukin-4 (IL-4) and IL-10 with a characteristic deficiency of IL-2 and interferon-γ (IFN-γ). This cytokine pattern is a representative of a mixed Th1/Th2 cell response in JRA. The CD3/CD28 costimulatory molecule was found to be a potent inducer of IL-4 and IL-10 secretion. PBMC-derived augmented IL-10 secretion was inhibited by exogenous Th1 cell type recombinant cytokines (IL-2, IL-12, and IFN-γ). Although IL-10 inhibits PBMC-induced proinflammatory IL-1α and tumor necrosis factor-α secretion, it had no major effect on IL-6 production. The finding of a distinctly enhanced mixed Th1/Th2 cell response cytokine (IL-4 and IL-10) pattern in JRA provides a framework for developing strategies for immunologic intervention in this rheumatic disorder in children.     

The complex role of interleukin-10 in autoimmunity

Interleukin-10 (IL-10) is a cytokine that has been tested in different clinical trials based on its ability to down regulate T helper 1-type responses, namely IFN-gamma secretion and activation of monocytes/macrophages. There is also evidence in different animal models, that IL-10 could be useful in controlling Th2-mediated inflammatory processes. However, IL-10 also displays immunostimulatory properties especially on B cells and activated CD8(+)T cells. These seemingly divergent effects may explain the apparent lack of activity or adverse effects observed after IL-10 treatment in several animal models or clinical trials. Nevertheless, the ability of IL-10 to induce the differentiation of a subset of regulatory CD4(+)T cells (Tr1) and the importance of IL-10 for the in vivo function of regulatory T cells tends to support the view of IL-10 as a crucial cytokine in the control of immune responses. In different in vivo models, these cells were shown to inhibit Th1 and Th2-type inflammatory responses through the secretion of IL-10. These Tr1 cells may thus be used in specific cellular therapy in order to deliver IL-10 precisely at the site of inflammation.     

ELISpot Displays a Better Detection over ELISA of T Helper (Th) 2-Type Cytokine-Production by Ex Vivo-Stimulated Antigen-Specific T Cells from Human Peripheral Blood

Detection of cytokines secreted by ex vivo antigen-stimulated peripheral blood mononuclear cells (PBMC) by ELISA is hampered by low frequencies of specific T cells and cellular receptor consumption. We investigated if ELISpot, measuring cytokine production at the single cell level, facilitated a better detection of the Th2 cytokines IL-4 and IL-5. PBMC from nickel-allergic (n = 31) and non-allergic subjects (n = 10) were stimulated with nickel or tetanus toxoid (TT) and cytokine production assessed by ELISpot and ELISA. By IL-4 ELISpot, 74% of the allergic and 0% control subjects responded to nickel and 56% of all subjects to TT. ELISA detected IL-4 after nickel stimulation only in 13% of the allergic subjects. Also detection of TT-induced IL-5 was improved by ELISpot with 54% subjects responding versus 24% in ELISA. In contrast, detection of nickel-induced IL-5 was more comparable between methods, most likely due to the 7-fold higher IL-5 production per cell in response to nickel versus TT. The low IL-5 response to TT was associated with a higher induction of the down-regulatory cytokine IL-10 by TT as compared to nickel (p < 0.001). Overall, ELISpot displayed a better detection of IL-4 as well as low intensity IL-5 responses thus emphasizing the importance of selecting suitable methods for the measurement of cytokine production ex vivo.     

Interleukin-4 inhibits cyclo-oxygenase-2 expression and prostaglandin E production by human mature dendritic cells

Interleukin-4 (IL-4) is considered the key cytokine for inducing T helper type 2 (Th2) cell differentiation, while interferon-gamma and IL-12 are pivotal cytokines for Th1 immune responses. Paradoxically, IL-4 has also been demonstrated to enhance IL-12 production by dendritic cells, suggesting an IL-4-dependent regulatory feedback of the Th1/Th2 system. In addition, prostaglandin E(2) (PGE(2)), a lipid mediator of inflammation, has been implicated in the enhancement of Th2-type responses acting directly on T and B lymphocytes. PGE(2) synthesis is dependent on the serial engagement of various enzymes, among which the inducible cyclo-oxygenase-2 (COX-2) exerts a critical role in monocytes and dendritic cells. In this study we demonstrate that IL-4 inhibits COX-2 gene expression and consequently prevents secretion of PGE(2) by mature human dendritic cells. We also show that PGE(2) does not regulate IL-12 and IL-10 production by dendritic cells in an autocrine fashion. Hence, we suggest that IL-4 may exploit an IL-12-independent regulatory feedback of the Th1/Th2 system through PGE(2) inhibition.     

Differential modulation of T helper type 1 (Th1) and T helper type 2 (Th2) cytokine secretion by prostaglandin E2 critically depends on interleukin-2

Prostaglandin E₂ (PGE₂) favors T helper type 2 (Th2)-like cytokine secretion profiles in murine and human CD4⁺ T cells by inhibiting the production of the Th1-associated cytokines interleukin-2 (IL-2) and interferon-γ (IFN-γ) and up-regulating the production of the Th2-associated cytokines IL-4 and IL-5 in a dose-dependent way. However, the potent inhibition of IL-2 production by PGE₂ seems to be in contrast with the simultaneous up-regulation of IL-4 and IL-5 production, because the induction of these cytokines requires IL-2. We, therefore, investigated to which extent the net modulatory effect of PGE₂ is determined by the availability of IL-2. To this aim, we examined the effects of PGE₂ on the cytokine secretion profiles of a panel of human Th0 clones upon stimulation via different activation pathways, resulting either in high or low IL-2 production. The differential modulation of Th1 and Th2 cytokines by PGE₂ was observed only upon modes of stimulation resulting in high IL-2 production. When IL-2 production was low, PGE₂ inhibited the secretion of all four cytokines. These different modulation patterns were directly related to the IL-2 availability, because (i) neutralizing antibody to IL-2 abrogated the up-regulatory effect of PGE₂ on IL-4 and IL-5 secretion in experiments with high endogenous IL-2 levels, (ii) lack of differential cytokine modulation by PGE₂ in conditions with low levels of endogenous IL-2 could be restored with exogenous IL-2, and (iii) cell viability was comparable in all conditions. These results demonstrate that the net modulatory effect of PGE₂ on the cytokine secretion profile of T cells critically depends on the availability of IL-2. Since this parameter varies with the experimental conditions and the T cell population studied, this finding may explain why certain immune responses may be either up- or down-regulated by PGE₂ under different conditions.     

Soluble CD23 potentiates interleukin-1-induced secretion of interleukin-6 and interleukin-1 receptor antagonist by human monocytes

The low-affinity receptor for IgE (CD23) is cleaved into biologically active soluble fragments (sCD23), some of which have been reported to exhibit pleiotropic activities. However, it is not known whether the sCD23 fragments contribute to the induction and/or regulation of pro-inflammatory cytokine production. In this study, this possibility was tested using interleukin (IL)-1-stimulated human whole blood as an ex vivo model of cytokine cascade production. We show that human recombinant 25-kDa sCD23 significantly enhanced the production of IL-6 in whole blood stimulated by IL-1, but had only little or no effect in the absence of IL-1. The potentiating effect of sCD23 was concentration dependent within the range of plasma levels occurring during various inflammatory processes in man. These results prompted us to study whether sCD23 and IL-1 together also enhance the production of regulating factors exhibiting anti-cytokine activities. Our data indicate that sCD23 augments the release of IL-1 receptor antagonist induced by IL-1. Finally, examining the effect of sCD23 on human peripheral monocytes stimulated by IL-1, we confirmed the capacity of sCD23 to potentiate cytokine production. We suggest that sCD23 can modulate monocyte functions, thereby contributing to the amplification and regulation of immune and inflammatory processes.     

Normal B cells fail to secrete interleukin-12

Interleukin-12 is a key regulatory cytokine produced by antigen-presenting cells (APC) which drives the development of interferon-γ (IFN-γ)-producing cells and promotes cell-mediated immunity. Following subcutaneous immunization with protein antigen in adjuvant, dendritic cells (DC) but not small nor large B cells in immune lymph nodes express antigenic complexes and secrete substantial amounts of bioactive IL-12 p75 upon antigen-specific interaction with T cells. We have analyzed secretion of IL-12 p40 and p75 by cell populations enriched in DC, macrophages or B cells in response to nonspecific stimulation or to interaction with antigen-specific CD4⁺ cells. These APC populations do not produce IL-12 constitutively but, upon stimulation with heat-fixed Staphylococcus aureus and IFN-γ, IL-12 p40 and p75 are secreted by DC and macrophages, whereas B cells fail to produce IL-12. B cells also fail to secrete IL-12 in response to stimulation with LPS and IFN-γ. Co-culture with CD4⁺ T hybridoma cells and antigen induces IL-12 secretion by DC. Up-regulation of IL-12 secretion by interaction with antigen-specific CD4⁺ T cells is abrogated by anti-class II monoclonal antibodies (mAb), by soluble CD40 molecules and by anti-CD40 ligand mAb, demonstrating a positive feedback between T cells and DC mediated by TCR-peptide/class II and by CD40-CD40 ligand interactions. Expression of class II and CD40 molecules is comparable in B cells and DC, and both APC types activate CD4⁺ T cells. Yet, even upon interaction with antigen-specific T cells, B cells fail to secrete IL-12. The capacity of B cells to present antigen but not to secrete IL-12 may explain their propensity to selectively drive T helper type 2 cell development.     

Human eosinophils constitutively express multiple Th1, Th2, and immunoregulatory cytokines that are secreted rapidly and differentially

Eosinophils are innate immune leukocytes implicated in the initiation and maintenance of type 2 immune responses, including asthma and allergy. The ability to store and rapidly secrete preformed cytokines distinguishes eosinophils from most lymphocytes, which must synthesize cytokine proteins prior to secretion and may be a factor in the apparent Th2 bias of eosinophils. Multiple studies confirm that human eosinophils from atopic or hypereosinophilic donors can secrete over 30 cytokines with a varying and often opposing immune-polarizing potential. However, it remains unclear whether all of these cytokines are constitutively preformed and available for rapid secretion from eosinophils in the circulation of healthy individuals or are restricted to eosinophils from atopic donors. Likewise, the relative concentrations of cytokines stored within eosinophils have not been studied. Here, we demonstrate that human blood eosinophils are not singularly outfitted with Th2-associated cytokines but rather, constitutively store a cache of cytokines with nominal Th1, Th2, and regulatory capacities, including IL-4, IL-13, IL-6, IL-10, IL-12, IFN-gamma, and TNF-alpha. We demonstrate further rapid and differential release of each cytokine in response to specific stimuli. As agonists, strong Th1 and inflammatory cytokines elicited release of Th2-promoting IL-4 but not Th1-inducing IL-12. Moreover, a large quantity of IFN-gamma was secreted in response to Th1, Th2, and inflammatory stimuli. Delineations of the multifarious nature of preformed eosinophil cytokines and the varied stimulus-dependent profiles of rapid cytokine secretion provide insights into the functions of human eosinophils in mediating inflammation and initiation of specific immunity.     

Interleukin-12 increases interleukin-4 production by established human ThO and Th2-like T cell clones

Interleukin (IL)-12 is a potent inducer of cell-mediated immunity: it favors the generation of interferon (IFN)-γ-producing T cells, increases IFN-γ production by T cells and natural killer cells and prevents the generation of a Th2 response in murine in vivo models. Nevertheless, the effects of IL-12 on an established Th2 response remain poorly documented. In the present paper, we analyzed the effect of IL-12 on the profile of lymphokines produced by established IL-4-producing Th0 and Th2-like human T cell clones (TCC) and by polyclonal T cells. We found that IL-12 (i) enhances, as previously reported, IFN-γ production by Th0 TCC and, to a smaller extent, by Th2-like TCC, (ii) increases the proliferation of Th0 and Th2-like TCC and (iii) unexpectedly, synergizes with T cell receptor-associated or nonspecific stimuli in increasing IL-4 production by these TCC. Thus, IL-12 potentiates the production of IFN-γ and also of IL-4 by established IL-4-producing TCC. Although IL-12 has been widely reported to induce a Th1 response and to prevent the development of a Th2 response in vivo, IL-12 may on the contrary potentiate an established Th2 response in humans.     

Relationship between HLA polymorphisms and gamma interferon and interleukin-10 cytokine production in healthy individuals after rubella vaccination

We studied the association between HLA alleles and rubella-specific gamma interferon (IFN-gamma) (Th1) and interleukin-10 (IL-10) (Th2) cytokine responses among 106 healthy children (ages, 14 to 17 years) previously immunized with two doses of rubella vaccine. Antibody titers and cytokine responses to rubella vaccination were not sex or age dependent. Several class I HLA-A (*0201, *2402, *6801) alleles were significantly associated with rubella vaccine-induced IFN-gamma secretion. Several class II HLA-DRB1 (*0101) and HLA-DQB1 (*0501) alleles were also suggestive of an association with IFN-gamma secretion. Alleles with potential associations with rubella-specific IL-10 production included HLA-A (*0201, *6801), HLA-B (*4901), and HLA-DRB1 (*1302). The class I A*0201 and A*6801 alleles were associated with both IFN-gamma and IL-10 secretion. These tentative associations need to be validated in larger studies with subjects of differing ethnicities. These results provide additional evidence that HLA genes may influence Th1- and Th2-specific cytokine response(s) following rubella immunization, which in turn can influence both cellular and humoral immune responses to rubella vaccination.     

Linked in vivo expression of soluble interleukin-4 receptor and interleukin-4 in murine schistosomiasis

Soluble interleukin-4 receptors (sIL-4R) are truncated IL-4R molecules that are secreted into biological fluids. To gain an insight into the mechanisms that control sIL-4R synthesis in vivo and their role in the regulation of immune responses, the expression and secretion of sIL-4R in mice infected with Schistosoma mansoni was studied. Splenocytes from infected animals responded to schistosomal antigen preparations with increased production of both IL-4 and sIL-4R. The synthesis of sIL-4R by spleen cells peaked at 8 weeks following infection and coincided with maximum levels of sIL-4R in serum and sIL-4R-specific mRNA in the liver of infected mice. The expression of IL-4-specific mRNA in the liver was different from that of IL-4R, reaching its peak approximately 2 weeks earlier. A relationship between sIL-4R production and the development and activation of Th2 cells was suggested by the findings that: (a) in vivo administration of anti-IL-4 antibodies (11B11) impaired the ability of splenic cells to secrete either IL-4 or sIL-4R; and (b) splenic cells from mice vaccinated with irradiated cercariae, which tend to develop much weaker Th2 responses than mice injected with live cercariae, expressed reduced levels of sIL-4R when challenged with schistosomal antigens. Moreover, a direct role for IL-4 in regulating the expression of sIL-4R was suggested by the ability of anti-IL-4 antibodies to inhibit sIL-4R synthesis in vitro. These data provide the first evidence demonstrating that the production of sIL-4R in vivo is up-regulated during immune responses, especially during those characterized by the development and activation of Th2 cells and IL-4 secretion. The association between sIL-4R and IL-4 syntheses is consistent with a potential role for sIL-4R in the regulation of IL-4 activity in vivo.