Anti-inflammatory response of IL-4, IL-10 and TGF-beta in patients with systemic inflammatory response syndrome

The systemic inflammatory response syndrome (SIRS) is an inflammatory process seen in association with a large number of clinical infective and non-infective conditions. The aim of this study was to investigate the role of anti-inflammatory cytokines such as interleukin-4 (IL-4), interleukin-10 (IL-10), and transforming growth factor-beta (TGF-beta). Serum levels of IL-4, IL-10 and TGF-beta were determined in 45 patients with SIRS: 38 patients had SIRS of infectious origin, whereas seven patients had non-infectious SIRS. Twenty healthy subjects were used as controls. Serum levels of IL-4, IL-10 and TGF-beta were determined by an immunoenzyme assay. A significant increase of IL-4 was observed in these patients at the time of diagnosis and 5 days later. In contrast, serum levels of IL-10 were not increased at the time of diagnosis, but a slight decrease was noted after 5 days. Serum levels of TGF-beta were not increased at time of diagnosis, and a slight increase was observed after 5 days. Serum levels of IL-4 were significantly higher in patients with infectious SIRS at the time of diagnosis, whereas no significant difference between infectious and non-infectious SIRS was noted for serum levels of IL-10 and TGF-beta at the time of diagnosis and 5 days later. During SIRS, serum levels of IL-4 were significantly increased with a significant correlation between IL-4 and mortality, and only levels of IL-4 were significantly increased in the SIRS caused by infectious stimuli.     

T cells enhance production of IL-18 by monocytes in response to an intracellular pathogen

We studied the effect of T cells on IL-18 production by human monocytes in response to Mycobacterium tuberculosis. Addition of activated T cells markedly enhanced IL-18 production by monocytes exposed to M. tuberculosis. This effect was mediated by a soluble factor and did not require cell-to-cell contact. The effect of activated T cells was mimicked by recombinant IFN-gamma and was abrogated by neutralizing Abs to IFN-gamma. IFN-gamma also enhanced the capacity of alveolar macrophages to produce IL-18 in response to M. tuberculosis, suggesting that this mechanism also operates in the lung during mycobacterial infection. IFN-gamma increased IL-18 production by increasing cleavage of pro-IL-18 to mature IL-18, as it enhanced caspase-1 activity but did not increase IL-18 mRNA expression. These findings suggest that activated T cells can contribute to the initial immune response by augmenting IL-18 production by monocytes in response to an intracellular pathogen.     

Histone deimination as a response to inflammatory stimuli in neutrophils

Posttranslational modifications, such as the deimination of arginine to citrulline by peptidyl arginine deiminase (PAD4), change protein structure and function. For autoantigens, covalent modifications represent a mechanism to sidestep tolerance and stimulate autoimmunity. To examine conditions leading to histone deimination in neutrophils, we used Abs that detect citrullines in the N terminus of histone H3. Deimination was investigated in human neutrophils and HL-60 cells differentiated into granulocytes. We observed rapid and robust H3 deimination in HL-60 cells exposed to LPS, TNF, lipoteichoic acid, f-MLP, or hydrogen peroxide, which are stimuli that activate neutrophils. Importantly, we also observed H3 deimination in human neutrophils exposed to these stimuli. Citrullinated histones were identified as components of extracellular chromatin traps (NETs) produced by degranulating neutrophils. In contrast, apoptosis proceeded without detectable H3 deimination in HL-60 cells exposed to staurosporine or camptothecin. We conclude that histone deimination in neutrophils is induced in response to inflammatory stimuli and not by treatments that induce apoptosis. Our results further suggest that deiminated histone H3, a covalently modified form of a prominent nuclear autoantigen, is released to the extracellular space as part of the neutrophil response to infections. The possible association of a modified autoantigen with microbial components could, in predisposed individuals, increase the risk of autoimmunity.     

Highly concentrated urine-purified Tac peptide fails to inhibit IL-2-dependent cell proliferation in vitro.

Tac peptide, i.e., the p55 chain of the human interleukin-2 receptor (IL-2R) complex, is detectable as a soluble from (sIL-2R) in normal sera and, at increased levels, in patients with different diseases. Since several immunological abnormalities are observed in most conditions associated with an increase in sIL-2R levels, a down-regulatory effect on IL-2-dependent functions has been postulated as a consequence of binding and functional block of IL-2 by the excess of sIL-2R. To test this hypothesis, we purified sIL-2R from the urine of a patient with hairy cell leukemia and investigated the possible inhibitory effect of this peptide on the in vitro IL-2-induced cell proliferation. The urine-purified molecule was detectable by the specific immunoassay utilized to measure the serum Tac peptide and was constructed by a single polypeptide of about 50 kDa which was able to bind IL-2. Experiments performed with the IL-2-dependent murine CTLL-2 cell line and with PHA-stimulated human peripheral blood mononuclear cells showed that the purified sIL-2R at concentrations up to about 300 nM was unable to block IL-2-dependent cell proliferation. According to these data, which can be explained by the low affinity for IL-2 of the p55 IL-2R chain, it seems unlikely that in vivo the soluble Tac peptide can exert a down regulatory effect on IL-2-induced phenomena through a functional block of IL-2.     

IL-18 gene polymorphisms affect IL-18 production capability by monocytes

We previously demonstrated a significant association between IL-18 gene polymorphism 105A/C and asthma. In this study, we investigated the relationship of IL-18 gene polymorphism to IL-18 production capability by monocytes. The frequency of gene polymorphisms including IL-18-105A/C and IL-18--137G/C was determined by PCR analyses. The IL-18 production by monocytes stimulated without or with LPS or A23187+PMA for 1day was measured by ELISA. The produced IL-18 spontaneously or in response to A23187+PMA by monocytes was significantly higher for volunteers with 105A/A genotype than with 105A/C genotype. Similarly, the production capability of IL-18 by monocytes from volunteers with -137G/G genotype was significantly higher than that with -137G/C genotype and significant linkage disequilibrium was observed between 105A/C and -137G/C polymorphism. Thus, the genetic capacity to produce more IL-18 in response to stimuli may affect the onset of asthma.     

Pancreatic glucagon fails to inhibit sham feeding in the rat

Rats were equipped with chronic gastric cannulas, and the effects of intraperitoneal injections of pancreatic glucagon on sham feeding, with cannulas open, and real feeding, with cannulas closed, were compared. Glucagon (100–2,500 μg/kg) suppressed cumulative food intake during real feeding tests 9–33%, but had no effect during sham feeding. Despite their increased food intakes, sham feeding rats took discrete meals terminated by the behavioral satiety sequence. In addition to not affecting total intake, glucagon failed to affect meal size, latency to rest, or intermeal interval during sham feeding. To investigate the possiblity that glucagon did not inhibit sham feeding because it did not elicit hyperglycemia, we measured hepatic vein blood glucose after glucagon injections in sham feeding rats: glucagon elicited marked hyperglycemia during sham feeding. Therefore, the absence of glucagon's satiety effect in sham feeding is not due to the lack of hepatic glycogenolysis and hyperglycemia. These results suggest that some mechanism other than hyperglycemia which normally accompanies food ingestion is necessary for glucagon to have a satiety effect.     

Different modes of IL-10 and TGF-beta to inhibit cytokine-dependent IFN-gamma production: consequences for reversal of lipopolysaccharide desensitization

LPS hyporesponsiveness is characterized by a diminished production of proinflammatory cytokines which can be caused by pretreatment with either LPS (=LPS desensitization) or the combination of the anti-inflammatory cytokines IL-10 and TGF-beta. However, the resulting hyporesponsive states differ regarding their reversibility by the IFN-gamma-inducing cytokine IL-12. Therefore, we aimed at studying the reasons for this differential IL-12 responsiveness of IFN-gamma-producing cells and its consequences for LPS hyporesponsiveness in more detail. In an in vitro IL-12/IL-18 responsiveness model, we demonstrated that IL-10, if permanently present, does not directly inhibit IL-12/IL-18 responsiveness in T/NK cells but indirectly interferes with IFN-gamma production in the presence of monocytes. In contrast, TGF-beta acted directly on IFN-gamma-producing cells by interfering with IL-12/IL-18 responsiveness. After removal of IL-10 but not of TGF-beta, LPS hyporesponsiveness can be reverted by IL-12/IL-18. Consequently, the addition of recombinant TGF-beta during LPS desensitization rendered PBMCs hyporesponsive to a reversal by IL-12/IL-18. Our data suggest that the persistence of IL-10 and the presence of TGF-beta determine the level of IFN-gamma inhibition and may result in different functional phenotypes of LPS desensitization and LPS hyporesponsiveness in vitro and in vivo.     

IL-21 effects on human IgE production in response to IL-4 or IL-13

In human atopic disease, IgE sensitizes the allergic response, while IgG4 is protective. Because IL-4 and IL-13 trigger switch recombination to both IgE and IgG4, additional agents must regulate the balance between these isotypes to influence susceptibility or tolerance to atopy. In this report, we define in vitro conditions leading to activation or inhibition of human IgE and IgG4 production by IL-21. IL-21 reduced IL-4-driven IgE synthesis by mitogen-stimulated human PBMC. IL-21 inhibition of human IgE production was not a direct effect on B cells, was not seen following B cell activation with IL-13, and was overcome by CD40 ligation. Neither IFN-gamma, IL-10, IL-12, CD40L expression, nor apoptosis was responsible for the inhibitory effect. In contrast, IL-21-stimulated secretion of IgG4 from PBMC. Our findings indicate that IL-21 may influence the production of both human IgE and IgG4, and thus contribute to the regulation of atopic reactions.     

Decreased response to IL-12 and IL-18 of peripheral blood cells in rheumatoid arthritis

Inflamed synovium of rheumatoid arthritis (RA) has been associated with a T helper (Th)1 cytokine profile but the blood situation remains to be clarified. We studied the differential IFN-gamma producing activity of peripheral blood mononuclear cells (PBMCs) from RA patients (RA-PBMCs) and from healthy controls (H-PBMCs) in response to IL-12 and IL-18. RA-PBMCs had a decreased IFN-gamma production in response to IL-12 and IL-18 when compared with H-PBMCs. RA-PBMCs activated with phytohemagglutinin and phorbol 12-myristate 13-acetate showed an increased sensitivity to IL-12 and IL-18, but still the RA-PBMC response was lower. IL-18 increased IL-12-stimulated IFN-gamma production from RA synovium cells obtained after collagenase digestion more effectively than that of RA- or H-PBMCs. A specific inhibitor of IL-18 bioactivity, IL-18-binding protein (IL-18BP), down-regulated IL-12-induced IFN-gamma production by RA- or H-PBMCs and had a remarkable effect on RA synovium cells. In conclusion, RA disease combines a polarized immune response with an active Th1 in inflamed joints and a reduced Th1 pattern in peripheral circulation.     

Decreased response to IL-12 and IL-18 of peripheral blood cells in rheumatoid arthritis

Inflamed synovium of rheumatoid arthritis (RA) has been associated with a T helper (Th)1 cytokine profile but the blood situation remains to be clarified. We studied the differential IFN-γ producing activity of peripheral blood mononuclear cells (PBMCs) from RA patients (RA-PBMCs) and from healthy controls (H-PBMCs) in response to IL-12 and IL-18. RA-PBMCs had a decreased IFN-γ production in response to IL-12 and IL-18 when compared with H-PBMCs. RA-PBMCs activated with phytohemagglutinin and phorbol 12-myristate 13-acetate showed an increased sensitivity to IL-12 and IL-18, but still the RA-PBMC response was lower. IL-18 increased IL-12-stimulated IFN-γ production from RA synovium cells obtained after collagenase digestion more effectively than that of RA- or H-PBMCs. A specific inhibitor of IL-18 bioactivity, IL-18-binding protein (IL-18BP), down-regulated IL-12-induced IFN-γ production by RA- or H-PBMCs and had a remarkable effect on RA synovium cells. In conclusion, RA disease combines a polarized immune response with an active Th1 in inflamed joints and a reduced Th1 pattern in peripheral circulation.     

IL-10 synergizes with IL-4 and transforming growth factor-beta to inhibit macrophage cytotoxic activity.

After activation with IFN-gamma, thioglycollate-elicited murine peritoneal macrophages kill schistosomula of Schistosoma mansoni in vitro by an L-arginine-dependent mechanism which involves the production of reactive nitrogen oxides (NO). In the present study we demonstrate that the regulatory cytokines IL-10, IL-4, and transforming growth factor-beta (TGF-beta) are potent inhibitors of this extracellular killing function of activated macrophages. Each cytokine was found to suppress killing of schistosomula in a dose-dependent fashion. The activity of IL-10 was not permanent, because subsequent treatment with additional IFN-gamma 2 to 6 h later reversed the inhibition of macrophage larval killing. More importantly, the combination of suboptimal levels of any two of these three cytokines was found to give a potent synergistic suppression of schistosomulum killing by IFN-gamma-treated macrophages. Similarly, IL-10, IL-4, or TGF-beta alone blocked the production of NO, and when used in combination these cytokines exhibited an enhanced inhibitory effect on nitrite production. Macrophage-mediated killing of schistosomula through the generation of NO has been shown previously to be a major effector mechanism of schistosome immunity. The results presented here suggest that the suppression of this mechanism by induction of the regulatory cytokines IL-10, IL-4, and TGF-beta, which are known to be produced during schistosome infection, may be an important strategy used by the parasite to evade macrophage-mediated immune destruction.     

Isolated peripheral T cells from GvHR recipients exhibit defective IL-2R expression, IL-2 production, and proliferation in response to activation stimuli

Graft-vs-host reactions (GvHR) following the injection of class I/II MHC disparate parental cells into unirradiated F1 recipient mice result in the development of marked immune dysfunction. Following negative selection using adherence and antibody and complement depletion, highly purified T cells were examined to determine their ability to undergo activation. Three weeks after GvHR initiation, unstimulated splenic T cells from GvHR mice displayed normal CD3 and IL-2R expression but elevated expression of class I MHC and Ly-6A/E antigens. Despite culture with normal F1 accessory cells, both CD4+ and CD8+ GvHR T cells exhibited low levels of proliferation to both Con A and anti-CD3 mAb. Although following exposure for 12 h to either of these stimuli, GvHR T cells expressed normal levels of IL-2R, expression was greatly decreased vs normal T cells between 24 and 48 h. In addition, at no timepoint was detectable IL-2 produced by GvHR T cells. Importantly, mixing experiments did not demonstrate detectable suppressive activity in the purified GvHR T cell subsets. GvHR T cells were also tested for their ability to respond to stimuli in the absence of any accessory cell population. These cells again did not proliferate to levels equivalent to normal T cells. Incubation with PMA and either cytokines (Con A supernatant, rIL-7) or anti-CD3 mAb resulted in only low levels of proliferation in GvHR T cells. Notably, at high ionomycin concentrations together with PMA, GvHR T cells did proliferate to equivalent levels as normal cells. However, with decreasing concentrations of ionophore, these cells failed to proliferate as well as normal cells. In total, these findings demonstrate that GvHR T cells are phenotypically and functionally distinct from normal T cells. The results suggest that GvHR T cells themselves may contribute to the well-characterized immune depression occurring in recipients undergoing GvHR.     

Yersinia enterocolitica evasion of the host innate immune response by V antigen-induced IL-10 production of macrophages is abrogated in IL-10-deficient mice.

The virulence-associated V Ag (LcrV) of pathogenic Yersinia species is part of the translocation apparatus, required to deliver antihost effector proteins (Yersinia outer proteins) into host cells. An orthologous protein (denoted as PcrV) has also been identified in the ExoS regulon of Pseudomonas aeruginosa. Additionally, it is known that LcrV is released by yersiniae into the environment and that LcrV causes an immunosuppressive effect when injected into mice. In this study, we demonstrate for the first time that rLcrV, but not PcrV, is capable of suppressing TNF-alpha production in zymosan A-stimulated mouse macrophages and the human monocytic Mono-Mac-6 cell line. The underlying mechanism of TNF-alpha suppression could be assigned to LcrV-mediated IL (IL)-10 production, because 1) LcrV induces IL-10 release in macrophages, 2) anti-IL-10 Ab treatment completely abrogated TNF-alpha suppression, and 3) TNF-alpha suppression was absent in LcrV-treated macrophages of IL-10-deficient (IL-10-/-) mice. The relevance of LcrV-mediated immunosuppression for the pathogenicity of yersiniae became evident by experimental infection of mice; in contrast to wild-type mice, IL-10-/- mice were highly resistant against Yersinia infection, as shown by lower bacterial load in spleen and liver, absent abscess formation in these organs, and survival.