Barrier function coordinately regulates epidermal IL-1 and IL-1 receptor antagonist mRNA levels

Disruption of the cutaneous permeability barrier increases mRNA levels for TNF, GM-CSF, IL-1 alpha, and IL-1 beta in the epidermis. We have hypothesized that the cytokines mediate the changes in lipid and DNA synthesis which occur following barrier disruption. To further characterize the cytokine response to barrier abrogation, we examined the levels of epidermal IL-1ra mRNA in two acute models and one chronic model in the hairless mouse. IL-1ra mRNA levels increased shortly after acute disruption of the barrier with acetone, reached a peak at 3-4 h after treatment, and returned to control levels by 8h. These changes in mRNA levels parallel those which occur for IL-1 alpha and beta. Furthermore, IL-1ra mRNA levels were elevated 5-fold and 4-fold, at 2.5 h and 4 h, respectively, following tape-stripping, a second acute model of barrier disruption. Finally, IL-1ra mRNA levels were elevated 2.5-fold in the epidermis of EFAD mice, which have a chronic barrier defect. Thus, the cutaneous response to barrier disruption includes mechanisms which increase IL-1 and IL-1ra mRNA levels in a coordinate manner. The net result provides a regulatory mechanism for controlling the biological effects of increased IL-1 production.     

Expression of the IL-12 receptor beta 1 and beta 2 subunits in human tuberculosis

To determine whether the Th1 response in tuberculosis correlated with IL-12R expression, we measured expression of the IL-12R beta 1 and IL-12R beta 2 subunits, as well as IL-12R beta 2 mRNA expression in tuberculosis patients and healthy tuberculin reactors. In tuberculosis patients, IFN-gamma production by Mycobacterium tuberculosis-stimulated PBMC was reduced, the percentages of T cells expressing IL-12R beta 1 and IL-12R beta 2 were significantly decreased, and IL-12R beta 2 mRNA expression was also markedly reduced. In contrast, in pleural fluid and lymph nodes at the site of disease in tuberculosis patients, in which IFN-gamma production is enhanced, IL-12R beta 2 mRNA expression was also increased. In M. tuberculosis-stimulated peripheral blood T cells from tuberculosis patients, anti-IL-10 and anti-TGF-beta enhanced IL-12R beta 1 and IL-12R beta 2 expression, and IFN-gamma production. In M. tuberculosis-stimulated peripheral blood T cells from healthy tuberculin reactors, recombinant IL-10 and TGF-beta reduced IL-12R beta 1 and IL-12R beta 2 expression, as well as IFN-gamma production. In combination with prior studies showing increased production of TGF-beta by blood monocytes from tuberculosis patients, this suggests that increased TGF-beta production is the underlying abnormality that reduces IL-12R beta 1 and IL-12R beta 2 expression in tuberculosis. Our findings provide evidence that IL-12R expression correlates well with IFN-gamma production in human tuberculosis, and that expression of IL-12R beta 1 and IL-12R beta 2 may play a central role in mediating a protective Th1 response.     

Comparison of the effects of interleukin-1 alpha, interleukin-1 beta and interferon-gamma-inducing factor on the production of interferon-gamma by natural killer

Interferon-gamma inducing factor (IGIF) is a recently identified cytokine which stimulates the production of interferon-gamma (IFN-gamma) by T cells and enhances natural killer (NK) cell cytolytic activity. Protein fold recognition, structure prediction and comparative modeling have revealed that IGIF is a member of the interleukin (IL)-1 cytokine family and has prompted the designation IL-1 gamma. Here we report functional similarities between members of the IL-1 family by comparing the effects of IL-1 alpha, IL-1 beta and IGIF on NK cell production of IFN-gamma. All three IL-1 types enhanced NK cell production of IFN-gamma when induced by IL-2 or IL-12, although at high concentrations (> 10 ng/ml), IGIF was five- to tenfold more potent than IL-1 alpha or IL-1 beta. This effect correlated with enhanced levels of mRNA for IFN-gamma when NK cells were stimulated with IGIF plus IL-12. In contrast to IL-12 and IL-2, the ability of IGIF to stimulate NK cell production of IFN-gamma was not increased by IL-1 alpha or IL-1 beta. The ability of IGIF to enhance IFN-gamma production was independent of the type I and type II IL-1 receptors or the IL-1R accessory protein. Together, these results identify IGIF as a potent stimulator of NK cell production of IFN-gamma and demonstrate that the effect of IGIF on NK cell production of IFN-gamma is similar to that of IL-1 alpha and IL-1 beta but distinct from that of IL-12.     

In vivo IL-12 production and IL-12 receptors beta1 and beta2 mRNA expression in the spleen are differentially up-regulated in resistant B6 and susceptible A/J mice during early blood-stage Plasmodium chabaudi AS malaria

As previously reported, blood-stage Plasmodium chabaudi AS malaria is lethal by days 10-12 postinfection in susceptible A/J mice that mount an early, predominantly Th2 response. In contrast, resistant C57BL/6 (B6) mice clear the infection by 4 wk with an early Th1 response. In this study, we analyzed in vivo production of IL-12, a potent Th1-inducing cytokine, during the first 5 days after P. chabaudi AS infection in these mice. By day 2, serum IL-12 p70 levels were significantly increased in B6 mice over basal levels and were also significantly higher compared with A/J mice that showed no significant changes in serum p70 levels after infection. Splenectomy of resistant B6 mice before infection demonstrated that the spleen is the major source of systemic IL-12 in these hosts. Splenic mRNA levels of both p40 and p35 were significantly higher in A/J mice; however, the ratios of p40/p35 mRNA levels were similarly up-regulated in both strains. Furthermore, B6 but not A/J mice showed significant up-regulation of splenic IL-12R beta2 mRNA over basal levels by days 3 and 4, coincident with sustained up-regulation of splenic IFN-gamma mRNA levels on days 3-5. However, IL-12R beta1 mRNA levels in the spleen were similarly up-regulated in both mouse strains by day 3. Taken together, these data suggest that high systemic IL-12 production, accompanied by an early and sustained up-regulation of both IL-12R beta1 and beta2 mRNA levels in the spleen, as occurs in resistant B6 mice, appears to preferentially induce protective Th1 responses against blood-stage malaria.     

Leukemia inhibitory factor modulates interleukin-1beta-induced activation of the hypothalamo-pituitary-adrenal axis

We have shown that leukemia inhibitory factor (LIF) is expressed in corticotroph cells and stimulates POMC gene expression and ACTH secretion in vivo and in vitro. We therefore examined the regulation of in vitro and in vivo pituitary LIF expression by cytokines known to stimulate the hypothalamo-pituitary-adrenal axis. In the corticotroph cell line AtT-20/D16v-F2, recombinant murine interleukin-1beta (IL-1beta; 0.1-10.0 ng/ml) caused a 5- to 10-fold increase in LIF messenger RNA (mRNA) levels. LIF mRNA expression was induced as early as 1 h, peaked at 2 h, and still persistently elevated above the baseline after 8 h. This effect of IL-1beta on LIF mRNA expression was abolished by preincubation with human IL-1 receptor antagonist (100 ng/ml) or antimurine IL-1beta antibody (10 microg/ml). Tumor necrosis factor-alpha (20 ng/ml) only modestly increased LIF mRNA, but was synergistic with IL-1beta (up to 2.5-fold). In contrast, IL-2 and IL-6 did not alter LIF mRNA. In C57BL/6 mice, i.p. injection of 100 ng IL-1beta increased plasma ACTH and corticosterone levels after 1 h (P < 0.02). In addition, pituitary LIF mRNA content was increased for up to 2 h in response to IL-1beta. In comparison to wild-type (+/+) B6D2F1 mice, LIF knockout mice with a deleted LIF gene (-/-) exhibited decreased plasma ACTH (631 +/- 61 vs. 376 +/- 50 pg/ml; P < 0.01) and corticosterone (783 +/- 85 vs. 433 +/- 51 ng/ml; P < 0.01) levels 1 h after i.p. IL-1beta administration. In conclusion, corticotroph LIF mRNA expression is specifically stimulated by IL-1beta and tumor necrosis factor-alpha. The attenuated hypothalamo-pituitary-adrenal response to IL-1beta in LIF knockout mice indicates that the effect of IL-1beta on ACTH secretion is modulated by LIF. Thus, LIF appears to function as an immune-neuroendocrine modulator signaling the hypothalamo-pituitary-adrenal axis.     

HIV-1 gp120 modulates hypothalamic cytokine mRNAs in vivo: implications to cytokine feedback systems

HIV-1-derived envelope glycoprotein 120 (gp120) may play an important role in HIV-1 neuropathology. Gp120 may act through mediators including proinflammatory cytokines. Here, we investigated the regulation of the IL-1 beta system [IL-1 beta, IL-1 receptor type I (IL-1RI), IL-1 receptor antagonist (IL-1Ra), IL-1 receptor accessory proteins (IL-1R AcP I and II)], TNF-alpha, TGF-alpha, and TGF-beta 1 mRNAs in the hypothalamus of Wistar rats in response to the chronic intracerebroventricular (ICV) microinfusion (via osmotic minipumps) of HIV-1 gp120 (100, 500, and 1000 ng/24 h for 72 h). Gp120 increased IL-1 beta, IL-1Ra, TNF-alpha, and TGF-beta 1 mRNAs. Gp120-induced cytokine mRNA profiles were highly intercorrelated in the same samples. Levels of IL-1RI, IL-1R AcP I and II, and TGF-alpha did not change significantly, and levels of GAPDH mRNA were constant. The data suggest potential cytokine-cytokine interactions with positive (IL-1 beta<-->TNF-alpha) and negative (IL-1Ra-->IL-1 beta; TGF-beta 1-->IL-1 beta/TNF-alpha) feedback in gp120 action. A dysregulation of the balance between stimulatory and inhibitory cytokine mechanisms may participate in the initiation, propagation, and/or aggravation of HIV-1 neuropathology.     

Mg2+ coordination in catalytic sites of F1-ATPase

Cytokines are hormone-like proteins which mediate and regulate inflammatory and immune responses. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) and inflammatory cytokines on regulation of interleukin-6 (IL-6) production by human gingival fibroblasts (HGF). The HGF cell lines used in this study, H-CL and F-CL, were established by the explant technique from healthy gingival tissue. Cultured cells were grown to confluency and incubated with various concentrations of LPS from Escherichia coli or Porphyromonas gingivalis or with the recombinant human cytokine tumor necrosis factor alpha (TNF-alpha), IL-1alpha, or IL-1beta. Culture supernatants were collected at various times and assessed for IL-6 production by enzyme-linked immunosorbent assay. Total RNA was isolated from the harvested cells and used to assess levels of IL-6 mRNA by the RNase protection assay. Both LPS preparations induced IL-6 production (1 to 4 ng of IL-6 per ml) by both HGF cell lines. Although TNF-alpha stimulated IL-6 production by HGF, > 10-fold-larger amounts were induced with IL-1alpha and IL-1beta. Furthermore, the addition of both IL-1alpha and TNF-alpha to cultured cells resulted in approximately 600- to 800-fold-higher levels of IL-6 than seen in control cultures, suggesting that these cytokines synergistically induced IL-6 production by HGF. IL-6 message in cultured cells was upregulated 20-fold by TNF-alpha, 1,000-fold by IL-1alpha and IL-1beta, and 1,400-fold by IL-1alpha plus TNF-alpha. IL-1alpha and TNF-alpha alone upregulate IL-6 production in a dose- and time-dependent fashion. The addition of IL-1alpha and TNF-alpha to cultured HGF cells resulted in a synergistic induction of IL-6 after 8 h of incubation and when greater than 10 pg of this combination per ml was used. Our studies show that inflammatory cytokines are hundreds of times more potent than LPS in stimulating IL-6 production by HGF.     

Inhibition of IL-10 expression by IFN-gamma up-regulates transcription of TNF-alpha in human monocytes

Stimulation of human monocytes with LPS induces expression of multiple cytokines, including TNF-alpha, IL-1 beta, IL-6, and IL-10, IL-10 expression is delayed relative to that of TNF-alpha, IL-1 beta, and IL-6. Furthermore, IL-10 feedback inhibits expression of TNF-alpha, IL-1 beta, and IL-6, thus providing an efficient autocrine mechanism for controlling proinflammatory cytokine production in monocytes. The Th1-type lymphokine, IFN-gamma, markedly up-regulates TNF-alpha production in monocytes. However, the precise mechanism by which IFN-gamma mediates this effect is unknown. We examined the effects of IFN-gamma on IL-10 expression in LPS-stimulated monocytes, and the relationship between IL-10 and TNF-alpha production in these cells. LPS stimulation induced rapid, ordered expression of multiple cytokines. Steady-state mRNA levels for TNF-alpha increased rapidly, reached maximal levels by 2 to 3 h poststimulation, and then declined sharply. IL-1 beta and IL-6 mRNA levels also increased markedly following stimulation with LPS, but decreased more slowly than did TNF-alpha. Down-regulation of mRNA for TNF-alpha, IL-1 beta, and IL-6 coincided with a delayed and more gradual increase in IL-10 mRNA levels. Furthermore, neutralization of IL-10 with anti-IL-10 Abs prolonged TNF-alpha mRNA expression, and significantly increased net TNF-alpha production. IFN-gamma suppressed expression of IL-10 mRNA and protein in a dose-dependent manner. Moreover, inhibition of IL-10 production correlated with a marked increase in both the magnitude and duration of TNF-alpha expression. Thus, potentiation of TNF-alpha production by IFN-gamma in monocytes is coupled to inhibition of endogenous IL-10 expression.     

Interferon-gamma potentiates interleukin (IL)-6 and tumor necrosis factor-alpha but not IL-1beta induced by endotoxin in the brain

Because interferon-gamma (IFN gamma) is present in the central nervous system during neurologic diseases associated with inflammation, its effect on endotoxin-induced cytokines was studied. Cerebrospinal fluid (CSF) and serum levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF alpha), their messenger RNA expression in brain areas (hypothalamus, hippocampus, and striatum) and in spleen were evaluated 2 and 8 h after endotoxin [lipopolysaccharide (LPS), 25 microg/rat i.c.v.], IFN gamma (2.5 microg/rat i.c.v.) or after their coadministration in rats. CSF and serum IL-1beta levels were increased by LPS alone and IFN gamma coadministration did not furtherly increase them. IFN gamma potentiated LPS effect on IL-6 and TNF alpha levels in both CSF and serum. LPS and IFN-gamma coadministration did not alter IL-1beta messenger RNA expression induced by LPS in brain areas and in spleen, but it potentiated that of IL-6 and TNF alpha. The present in vivo data show that i.c.v. coadministration of LPS and IFN gamma results in a potentiation of cytokine production (IL-6 and TNF alpha) which may trigger a cascade of events relevant to neurodegenerative processes. This action is independent of IL-1beta because the production of this cytokine is not altered by IFN gamma treatment.     

Localization of mRNA for inflammatory cytokines in radicular cyst tissue by in situ hybridization, and induction of inflammatory cytokines by human gingival fibroblasts in response to radicular cyst contents

The expression of mRNA encoding the inflammatory cytokines interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor alpha(TNF-alpha) have been examined in radicular cysts by in situ hybridization. Furthermore, the biological activity of the contents of radicular cysts (RCC) has been assayed by adding extracts of RCC to cultured human gingival fibroblasts (HGFs) and analyzing the culture medium for the release of inflammatory cytokines. In the epithelial layer, keratinocytes expressed all cytokine mRNAs examined at various levels. Basal layer cells expressed mRNA for each cytokine. In the subepithelial granulation tissue of the cysts, fibroblasts and macrophages expressed mRNA for IL-6, IL-8, IL-1beta and TNF-alpha mRNA at varying levels; especially clear expression of TNF-alpha and IL-1beta mRNA was detected on macrophages. The infiltrating lymphoid cells, largely composed of T cells and plasma cells, expressed these cytokine mRNAs, especially those encoding IL-6 and IL-8, at various levels. In vitro analysis indicated dose-dependent release of both IL-6 and IL-8 by HGFs in response to RCC. After heating to 100 degrees C for 10 min, RCC almost completely failed to stimulate IL-6 release from HGFs. Furthermore, anti-IL-1beta antibody (neutralization test) did not prevent the stimulation of IL-6 release by RCC. Significant amounts of IL-6 and IL-8 were detected in RCC in two cases, and a trace amount of IL-1beta was detected in one case. This study demonstrated the wide expression of mRNA encoding inflammatory cytokines in radicular cyst tissues, and RCC itself was capable of stimulating IL-6 and IL-8 production from HGFs.     

IL-15 enhances the response of human gamma delta T cells to nonpeptide [correction of nonpetide] microbial antigens

Human gamma delta T cells have the ability to rapidly expand and produce IFN-gamma in response to nonpeptide Ags of microbial pathogens, in particular a class of compounds known as the prenyl phosphates. We investigated the ability of IL-15, a T cell growth factor, to modulate prenyl phosphate-induced gamma delta T cell proliferation and cytokine production. IL-15 significantly enhanced the expansion of gamma delta T cells in the peripheral blood after stimulation in vitro with isopentenyl pyrophosphate. Moreover, using gamma delta T cell clones, we determined that IL-15-induced T cell proliferation was dependent on the IL-2R beta chain but not the IL-2R alpha chain. We therefore studied the IL-15R alpha chain expression in human gamma delta T cells in the presence or absence of nonpeptide Ags. We found IL-15R alpha mRNA expression in IL-15-stimulated and Ag-stimulated human gamma delta T cells but not in resting gamma delta T cells. Although IL-15 itself had little effect on the production of IFN-gamma, IL-15 plus IL-12 acted synergistically to augment IFN-gamma production by gamma delta T cells. Moreover, we showed that this increase in IFN-gamma could be explained by the dual activation of STAT1 and STAT4 by IL-15 and IL-12, respectively. Taken together, these results suggest that IL-15 may contribute to activation of human gamma delta T cells in the immune response to microbial pathogens.