Selective regulation of IL-10 signaling and function by zymosan

Balanced activity of pro- and anti-inflammatory cytokines during innate immune responses is required to allow effective host defense while avoiding tissue damage and autoimmunity. Induction of cytokine production after recognition of pathogen-associated molecular patterns (PAMPs) by innate immune cells has been well demonstrated, but modulation of cytokine function by PAMPs is not well understood. In this study we show that stimulation of macrophages with zymosan, which contains PAMPs derived from yeast, rapidly extinguished macrophage responses to IL-10, a suppressive cytokine that limits inflammatory tissue damage but also compromises host defense. The mechanism of inhibition involved protein kinase Cbeta and internalization of IL-10R, and was independent of TLR2 and phagocytosis. Inhibition of IL-10 signaling and function required direct contact with zymosan, and cells in an inflammatory environment that had not contacted zymosan remained responsive to the paracrine activity of zymosan-induced IL-10. These results reveal a mechanism that regulates IL-10 function such that antimicrobial functions of infected macrophages are not suppressed, but the activation of surrounding noninfected cells and subsequent tissue damage are limited. The fate of individual cells in an inflammatory microenvironment is thus specified by dynamic interactions among host cells, microbes, and cytokines that determine the balance between protection and pathology.     

Prostaglandin E2 promotes IL-4-induced IgE and IgG1 synthesis

PG of the E series are generally known to suppress immune responses, however, we have found that PGE synergizes with IL-4 to induce IgE and IgG1 production in LPS-stimulated murine B lymphocytes. PGE2 and PGE1 (10(-6) to 10(-8) M) significantly increase IgE and IgG1 production (up to 26-fold) at all concentrations of IL-4 tested. In addition to its effects on IgE and IgG1, PGE also causes a significant decrease in IgM and IgG3 synthesis, suggesting that PGE may promote IL-4-induced class switching. The specificity of the E series PG effect is demonstrated by the fact that PGF2 alpha (10(-6) M) does not alter production of any of these isotypes. Because PGE can mediate its effects through cAMP in some cases, we investigated the importance of cAMP levels in regulation of isotype expression. Other agents that increase intracellular cAMP levels (cholera toxin and dibutyryl cAMP) were assessed for their ability to regulate isotype differentiation. Cholera toxin (100 pg/ml) and dibutyryl cAMP (100 microM) significantly enhanced IgE and IgG1 production and diminished IgM and IgG3 synthesis. We also show that PGE and cholera toxin elevate intracellular cAMP in B lymphocytes in a dose-dependent manner. In contrast, PGF2 alpha (10(-6) M) and the B subunit of cholera toxin (100 pg/ml) did not increase cAMP and did not regulate the isotype of Ig produced, reiterating the importance of cAMP in enhancing isotype differentiation. Although PGE is known to inhibit a number of immune responses, our data show that it is not always inhibitory. PGE may play a role in atopy in vivo where PGE-secreting cells such as macrophages, follicular dendritic cells, and fibroblasts can promote IgE synthesis. This research emphasizes the importance of PGE in regulation of the humoral immune response and adds a new stimulatory action to the repertoire of known PGE effects.     

Prostaglandin E(2) produced by the lung augments the effector phase of allergic inflammation

Elevated PGE(2) is a hallmark of most inflammatory lesions. This lipid mediator can induce the cardinal signs of inflammation, and the beneficial actions of nonsteroidal anti-inflammatory drugs are attributed to inhibition of cyclooxygenase (COX)-1 and COX-2, enzymes essential in the biosynthesis of PGE(2) from arachidonic acid. However, both clinical studies and rodent models suggest that, in the asthmatic lung, PGE(2) acts to restrain the immune response and limit physiological change secondary to inflammation. To directly address the role of PGE(2) in the lung, we examined the development of disease in mice lacking microsomal PGE(2) synthase-1 (mPGES1), which converts COX-1/COX-2-derived PGH(2) to PGE(2). We show that mPGES1 determines PGE(2) levels in the naive lung and is required for increases in PGE(2) after OVA-induced allergy. Although loss of either COX-1 or COX-2 increases the disease severity, surprisingly, mPGES1(-/-) mice show reduced inflammation. However, an increase in serum IgE is still observed in the mPGES1(-/-) mice, suggesting that loss of PGE(2) does not impair induction of a Th2 response. Furthermore, mPGES1(-/-) mice expressing a transgenic OVA-specific TCR are also protected, indicating that PGE(2) acts primarily after challenge with inhaled Ag. PGE(2) produced by the lung plays the critical role in this response, as loss of lung mPGES1 is sufficient to protect against disease. Together, this supports a model in which mPGES1-dependent PGE(2) produced by populations of cells native to the lung contributes to the effector phase of some allergic responses.     

Conformational changes mediate interleukin-10 receptor 2 (IL-10R2) binding to IL-10 and assembly of the signaling complex

Interleukin-10 receptor 2 (IL-10R2) is a critical component of the IL-10.IL-10R1.IL-10R2 complex which regulates IL-10-mediated immunomodulatory responses. The ternary IL-10 signaling complex is assembled in a sequential order with the IL-10.IL-10R1 interaction occurring first followed by engagement of the IL-10R2 chain. In this study we map the IL-10R2 binding site on IL-10 using surface plasmon resonance and cell-based assays. Critical IL-10R2 binding residues are located in helix A adjacent to the previously identified IL-10R1 recognition surface. Interestingly, IL-10R2 binding residues located in the N-terminal end of helix A exhibit large structural differences between unbound cIL-10 and cIL-10.IL-10R1 crystal structures. This suggests IL-10R1-induced conformational changes regulate IL-10R2 binding and assembly of the ternary IL-10.IL-10R1.IL-10R2 complex. The basic mechanistic features of the assembly process are likely shared by six additional class-2 cytokines (viral IL-10s, IL-22, IL-26, IL-28A, IL28B, and IL-29) to promote IL-10R2 binding to six additional receptor complexes. These studies highlight the importance of structure in regulating low affinity protein-protein interactions and IL-10 signal transduction.     

Prostaglandin E2 (PGE2) differentially regulates the production of IL-2 and IL-3 by murine immune T-cells.

Eicosanoids are important mediators of inflammation, and have been shown to have potent, and usually suppressive immunoregulatory activities. In the paper, we have examined the role of prostaglandin (PGE2) production in the regulation of two cytokines, IL-2 and IL-3, which both play a key role in contact sensitivity and delayed type hypersensitivity reactions. In agreement with previous studies, we demonstrate that prostaglandins down-regulate IL-2 production in the system. Unexpectedly, however, IL-3 levels are enhanced in the presence of the prostaglandin PGE2 and conversely, are inhibited by treatment with aspirin, a potent inhibitor of prostaglandin metabolism. The implications of this result in terms of the immunoregulatory role of PGs will be discussed.     

Prostaglandin E2 selectively inhibits human CD4+ T cells secreting low amounts of both IL-2 and IL-4

PGE2 is a potent inflammatory mediator with profound immune regulatory actions. The present study examined the effects of PGE2 on the activation/proliferation of CD4+ T cells using 37 cloned CD4+ T cell lines. Ten T cell clones sensitive to PGE2 and 10 T cell clones resistant to PGE2, as measured by proliferation in response to anti-CD3 Ab, were selected for comparison. It was found that the PGE2-sensitive T cells were characterized by low production (<200 pg/ml) of both IL-2 and IL-4, while PGE2-resistant T cells secreted high levels (>1000 pg/ml) of IL-2, IL-4, or both. The roles of IL-2 and IL-4 were confirmed by the finding that addition of exogenous lymphokines could restore PGE2-inhibited proliferation, and PGE2-resistant Th1-, Th2-, and Th0-like clones became PGE2 sensitive when IL-2, IL-4, or both were removed using Abs specific for the respective lymphokines. In addition, we showed that the CD45RA expression in PGE2-sensitive T cells was significantly lower than that in PGE2-resistant cells (mean intensity, 1.2 +/- 0.6 vs 7.8 +/- 5.7; p = 0.001). In contrast, CD45RO expression in PGE2-sensitive T cells was significantly higher that that in PGE2-resistant cells (mean intensity, 55.7 +/- 15.1 vs 33.4 +/- 12.9; p = 0.02). In summary, PGE2 predominantly suppressed CD45RA-RO+ CD4+ T cells with low secretion of both IL-2 and IL-4.     

Tyk2 negatively regulates adaptive Th1 immunity by mediating IL-10 signaling and promoting IFN-gamma-dependent IL-10 reactivation

The Jak, Tyk2, is activated in response to IL-12 and IFN-alphabeta and promotes IFN-gamma production by Th1-type CD4 cells. Mice deficient in Tyk2 function have been previously shown to be resistant to autoimmune arthritis and septic shock but are acutely susceptible to opportunistic pathogens such as Toxoplasma gondii. In this study, we show that Tyk2, in addition to mediating the biological effects of IL-12 and IFN-alphabeta, is an important regulator for the signaling and expression of the immunosuppressive cytokine IL-10. In the absence of Tyk2, Ag-reactive CD4 cells exhibit impaired IL-10 synthesis following rechallenge of T. gondii vaccine-primed mice. The impaired IL-10 reactivation leads to unopposed antimicrobial effector mechanisms which results in a paradoxically superior protection of immune Tyk2(-/-) mice against virulent T. gondii challenge. We further demonstrate that Tyk2 indirectly controls CD4 IL-10 reactivation by signaling for maximal IFN-gamma secretion. The unexpected role of IFN-gamma in mediating IL-10 reactivation by Th1 cells provides compelling evidence that conditions driving Th1 responses establish a negative feedback loop, which will ultimately lead to its autoregulation. Thus, Tyk2 can be viewed as a dual-function Jak, mediating both pro and anti-inflammatory cytokine responses.     

Pulmonary overexpression of IL-10 augments lung fibrosis and Th2 responses induced by silica particles

Chronic inflammation and proinflammatory cytokines as well as T helper type 2 (Th2) cytokines have been involved in the pathogenesis of pulmonary injury and lung fibrosis. The actual role of IL-10 in lung fibrosis is still unclear because this cytokine has been identified as Th2 but possesses strong anti-inflammatory properties. To better dissect the potential role of IL-10 in silica-induced lung fibrosis, IL-10 was overexpressed in the lung of mice by adenoviral gene transfer during the inflammatory (administered at day -1) or the fibrotic (administered at day +30) stages of the disease. Pulmonary overexpression of IL-10 during both silica-induced lung inflammation and fibrosis exacerbated the fibrotic lesions as estimated by the measurement of hydroxyproline and other biochemical and histological markers. Increased expression of IL-10 significantly enhanced the number of lung lymphocytes and bronchoalveolar lavage fluid IgG1 but not IgG2a levels, indicating the induction of a Th2-like immune response. In addition, the production of the profibrotic Th2 cytokines IL-4 and IL-13 was also significantly increased upon IL-10 overexpression. No difference in transforming growth factor-beta or PGE(2) production was noted after adenoviral IL-10 treatment of silica-treated mice. Together, these data indicate that the increased expression of IL-10 significantly contributed to silica-induced lung fibrosis by exacerbating the Th2 response and the production of the profibrotic cytokines IL-4 and IL-13.     

IL-10 reduces Th2 cytokine production and eosinophilia but augments airway reactivity in allergic mice

Mononuclear phagocytes can interact with mesenchymal cells and extracellular matrix components that are crucial for connective tissue rearrangement. We asked whether blood monocytes can alter matrix remodeling mediated by human lung fibroblasts cultured in a three-dimensional collagen gel. Blood monocytes from healthy donors (>95% pure) were cast into type I collagen gels that contained lung fibroblasts. Monocytes in coculture inhibited the fibroblast-mediated gel contractility in a time- and concentration-dependent manner. The concentration of PGE(2), a well-known inhibitor of gel contraction, was higher (P < 0.01) in media from coculture; this media attenuated fibroblast gel contraction, whereas conditioned media from either cell type cultured alone did not. Three-dimensional cultured monocytes responded to conditioned media from cocultures by producing interleukin-1beta and tumor necrosis factor-alpha, whereas fibroblasts increased synthesis of PGE(2). Antibodies to interleukin-1beta and tumor necrosis factor-alpha blocked the monocyte inhibitory effect and reduced the amount of PGE(2) produced. The ability of monocytes to block the fibroblast contraction of matrix may be an important mechanism in regulating tissue remodeling.     

Prostaglandin E2 upregulates EGF-stimulated signaling in mitogenic pathways involving Akt and ERK in hepatocytes

Prostaglandins (PGs) such as PGE2 enhance proliferation in many cells, apparently through several distinct mechanisms, including transactivation of the epidermal growth factor (EGF) receptor (EGFR) as well as EGFR-independent pathways. In this study we found that in primary cultures of rat hepatocytes PGE2 did not induce phosphorylation of the EGFR, and the EGFR tyrosine kinase blockers gefitinib and AG1478 did not affect PGE2-stimulated phosphorylation of ERK1/2. In contrast, PGE2 elicited EGFR phosphorylation and EGFR tyrosine kinase inhibitor-sensitive ERK phosphorylation in MH1C1 hepatoma cells. These findings suggest that PGE2 elicits EGFR transactivation in MH1C1 cells but not in hepatocytes. Treatment of the hepatocytes with PGE2 at 3 h after plating amplified the stimulatory effect on DNA synthesis of EGF administered at 24 h and advanced and augmented the cyclin D1 expression in response to EGF in hepatocytes. The pretreatment of the hepatocytes with PGE2 resulted in an increase in the magnitude of EGF-stimulated Akt phosphorylation and ERK1/2 phosphorylation and kinase activity, including an extended duration of the responses, particularly of ERK, to EGF in PGE2-treated cells. Pertussis toxin abolished the ability of PGE2 to enhance the Akt and ERK responses to EGF. The results suggest that in hepatocytes, unlike MH1C1 hepatoma cells, PGE2 does not transactivate the EGFR, but instead acts in synergism with EGF by modulating mitogenic mechanisms downstream of the EGFR. These effects seem to be at least in part G(i) protein-mediated and include upregulation of signaling in the PI3K/Akt and the Ras/ERK pathways.     

Prostaglandin E2 negatively regulates AMP-activated protein kinase via protein kinase A signaling pathway

We investigated possible involvement of prostaglandin (PG) E2 in regulation of AMP-activated protein kinase (AMPK). When osteoblastic MG63 cells were cultured in serum-deprived media, Thr-172 phosphorylation of AMPK alpha-subunit was markedly increased. Treatment of the cells with PGE2 significantly reduced the phosphorylation. Ser-79 phosphorylation of acetyl-CoA carboxylase, a direct target for AMPK, was also reduced by PGE2. On the other hand, PGE2 reciprocally increased Ser-485 phosphorylation of the alpha-subunit that could be associated with inhibition of AMPK activity. These effects of PGE2 were mimicked by PGE2 receptor EP2 and EP4 agonists and forskolin, but not by EP1 and EP3 agonists, and the effects were suppressed by an adenylate cyclase inhibitor SQ22536 and a protein kinase A inhibitor H89. Additionally, the PGE2 effects were duplicated in primary calvarial osteoblasts. Together, the present study demonstrates that PGE2 negatively regulates AMPK activity via activation of protein kinase A signaling pathway.     

Prostaglandin E precursor fatty acids inhibit human IL-2 production by a prostaglandin E-independent mechanism

Essential fatty acids, from which PG derive, can participate in development and regulation of immune responses and have been shown to suppress inflammation and tissue injury in animal models. In this report, we investigate the effects of the immediate (DGLA, precursor to PGE1), arachidonic acid (AA, PGE precursors, dihomogamma linolenic acid (DGLA, precursor to PGE1), arachidonic acid (AA, precursor to PGE2), and eicosapentaenoic acid (EPA, precursor to PGE3) on IL-2 production by PHA-stimulated human PBMC. DGLA and AA inhibited IL-2 production in a dose-dependent manner: half-maximal inhibition was obtained by using the fatty acids at the dose of 10 micrograms/ml without significant effects on cell viability. EPA inhibited IL-2 production by PBMC of only some donors. Incubation of cells in the presence of oleic, stearic, and palmitic acids, which are not PG precursors, did not affect mitogen-induced IL-2 production. A progressive increase in incorporation of DGLA into cellular lipids was observed over a 48-h incubation period. IL-2 production was reduced also when PBMC were pretreated overnight with DGLA or AA and washed before exposure to PHA. Whereas addition of the cyclo-oxygenase inhibitor, indomethacin, at the time of mitogenic stimulation led to increased IL-2 production and prevented mitogen- and fatty acid-induced increases in PGE release, it had no significant effect on the capacity of the fatty acids to suppress IL-2 production. Time course experiments showed that DGLA and AA inhibited IL-2 production even at times of minimal or no PGE release by the treated cultures. Moreover, DGLA and AA inhibited IL-2 production by the human leukemia T cell line Jurkat which, when appropriately induced, is able to release high levels of IL-2 in the absence of accessory cells and measurable PGE production. Taken together, these data indicate that essential fatty acids inhibit IL-2 production directly without conversion into their cyclo-oxygenase pathway products, and suggest that human lymphocyte function may be altered profoundly by small changes in their fatty acid profile.     

Prostaglandin E2 on the electroencephalogram

Present study was prompted by the report from another center on the occasional occurrence of convulsions and abnormal electroencephalogram (E.E.G.) patterns in women aborted with intraamniotic prostaglandin F2α (PGF2α). Fifty four subjects were investigated by means of an E.E.G. taken before and after initiation of PGE2 administration. They included pregnant and non-pregnant patients, nearly half (23) of whom were known epileptics. One seizure was observed during PG administration in a man with daily psychomotor attacks who had not taken his anticonvulsants on the day the test was performed. PGE2 caused no alteration of the E.E.G. in subjects with a normal control tracing; in those with an abnormal E.E.G., a deterioration was seen in one and an improvement in three. It is concluded that PGE2 is not epileptogenic at doses required for termination of pregnancy.     

Prostaglandin E2 on the electroencephalogram

Present study was prompted by the report from another center on the occasional occurrence of convulsions and abnormal electroencephalogram (E.E.G.) patterns in women aborted with intraamniotic prostaglandin F2α (PGF2α). Fifty four subjects were investigated by means of an E.E.G. taken before and after initiation of PGE2 administration. They included pregnant and non-pregnant patients, nearly half (23) of whom were known epileptics. One seizure was observed during PG administration in a man with daily psychomotor attacks who had not taken his anticonvulsants on the day the test was performed. PGE2 caused no alteration of the E.E.G. in subjects with a normal control tracing; in those with an abnormal E.E.G., a deterioration was seen in one and an improvement in three. It is concluded that PGE2 is not epileptogenic at doses required for termination of pregnancy.