Cloning and characterization of a binding subunit of the interleukin 13 receptor that is also a component of the interleukin 4 receptor.

Interleukins 4 (IL-4) and 13 (IL-13) have been found previously to share receptor components on some cells, as revealed by receptor cross-competition studies. In the present study, the cloning is described of murine NR4, a previously unrecognized receptor identified on the basis of sequence similarity with members of the hemopoietin receptor family. mRNA encoding NR4 was found in a wide range of murine cells and tissues. By using transient expression in COS-7 cells, NR4 was found to encode the IL-13 receptor alpha chain, a low-affinity receptor capable of binding IL-13 but not IL-4 or interleukins 2, -7, -9, or -15. Stable expression of the IL-13 receptor alpha chain (NR4) in CTLL-2 cells resulted in the generation of high-affinity IL-13 receptors capable of transducing a proliferative signal in response to IL-13 and, moreover, led to competitive cross-reactivity in the binding of IL-4 and IL-13. These results suggest that the IL-13 receptor alpha chain (NR4) is the primary binding subunit of the IL-13 receptor and may also be a component of IL-4 receptors.

Similarities and differences in signal transduction by interleukin 4 and interleukin 13: analysis of Janus kinase activation.

The cytokines interleukin (IL) 4 and IL-13 induce many of the same biological responses, including class switching to IgE and induction of major histocompatibility complex class II antigens and CD23 on human B cells. It has recently been shown that IL-4 induces the tyrosine phosphorylation of a 170-kDa protein, a substrate called 4PS, and of the Janus kinase (JAK) family members JAK1 and JAK3. Because IL-13 has many functional effects similar to those of IL-4, we compared the ability of IL-4 and IL-13 to activate these signaling molecules in the human multifactor-dependent cell line TF-1. In this report we demonstrate that both IL-4 and IL-13 induced the tyrosine phosphorylation of 4PS and JAK1. Interestingly, although IL-4 induced the tyrosine phosphorylation of JAK3, we did not detect JAK3 phosphorylation in response to IL-13. These data suggest that IL-4 and IL-13 signal in similar ways via the activation of JAK1 and 4PS. However, our data further indicate that there are significant differences because IL-13 does not activate JAK3.

CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages

It has been demonstrated that CD8+ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4+ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8+ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8+ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8+ T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8+ T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8+ T-cell supernatants; and (iii) the CD8+ T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8+ T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8+ T-cell-derived HIV-suppressor factors.

Interleukin 18 stimulates HIV type 1 in monocytic cells

The cytokine interleukin (IL) 18 (formerly interferon γ-inducing factor) induces the T helper type 1 response. In the present studies, IL-18 increased HIV type 1 (HIV-1) production from 5- to 30-fold in the chronically infected U1 monocytic cell line. Inhibition of tumor necrosis factor (TNF) activity by the addition of TNF-binding protein reduced IL-18-stimulated HIV-1 production by 48%. In the same cultures, IL-18-induced IL-8 was inhibited by 96%. Also, a neutralizing anti-IL-6 mAb reduced IL-18-induced HIV-1 by 63%. Stimulation of U1 cells with IL-18 resulted in increased production of IL-6, and exogenous IL-6 added to U1 cells increased HIV-1 production 4-fold over control. A specific inhibitor of the p38 mitogen-activated protein kinase reduced IL-18-induced HIV-1 by 73%, and a 50% inhibition was observed at 0.05 μM. In the same cultures, IL-8 was inhibited by 87%. By gel-shift and supershift analyses, increased binding activity of the transcription factor NF-κB was measured in nuclear extracts from U1 cells 1 h after exposure to IL-18. These results demonstrate induction of HIV-1 by IL-18 in a monocyte target associated with an intermediate role for TNF and IL-6, activation of p38 mitogen-activated protein kinase, and nuclear translocation of NF-κB.

Interleukin 9: A candidate gene for asthma

Asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of atopy and bronchial hyperresponsiveness. Recent studies localized a major gene for asthma to chromosome 5q31-q33 in humans. Thus, this segment of the genome represents a candidate region for genes that determine susceptibility to bronchial hyperresponsiveness and atopy in animal models. Homologs of candidate genes on human chromosome 5q31-q33 are found in four regions in the mouse genome, two on chromosome 18, and one each on chromosomes 11 and 13. We assessed bronchial responsiveness as a quantitative trait in mice and found it linked to chromosome 13. Interleukin 9 (IL-9) is located in the linked region and was analyzed as a gene candidate. The expression of IL-9 was markedly reduced in bronchial hyporesponsive mice, and the level of expression was determined by sequences within the qualitative trait locus (QTL). These data suggest a role for IL-9 in the complex pathogenesis of bronchial hyperresponsiveness as a risk factor for asthma.

Molecular cloning, sequence, expression, and processing of the interleukin 16 precursor

Interleukin 16 (IL-16) has been shown to function as chemoattractant factor, as a modulator of T-cell activation, and as an inhibitor of immunodeficiency virus replication. The recent identification of inconsistencies in published IL-16 cDNA nucleotide sequences led to the proposal that IL-16 is synthesized in the form of a large precursor protein (pro-IL-16). To identify the true transcriptional start of the IL-16 mRNA rapid amplification of cDNA ends methods were applied. The complete pro-IL-16 cDNA was subsequently molecularly cloned, sequenced, and expressed in COS-7 cells. We report here that pro-IL-16 is most likely synthesized as a 67-kDa protein and is encoded from a major 2.6-kb transcript. Recombinant pro-IL-16 polypeptides are specifically cleaved in lysates of CD8(+) cells, suggesting that the naturally secreted bioactive form of IL-16 is smaller than the originally published 130 amino acids fragment. Moreover, in contrast to other interleukins such as IL-15, IL-16 mRNA expression is almost exclusively limited to lymphatic tissues underlining the potential of IL-16 as an immune regulatory molecule.

Lipophosphoglycan from Leishmania suppresses agonist-induced interleukin 1β gene expression in human monocytes via a unique promoter sequence

Leishmania are parasites that survive within macrophages by mechanism(s) not entirely known. Depression of cellular immunity and diminished production of interleukin 1β (IL-1β) and tumor necrosis factor α are potential ways by which the parasite survives within macrophages. We examined the mechanism(s) by which lipophosphoglycan (LPG), a major glycolipid of Leishmania, perturbs cytokine gene expression. LPG treatment of THP-1 monocytes suppressed endotoxin induction of IL-1β steady-state mRNA by greater than 90%, while having no effect on the expression of a control gene. The addition of LPG 2 h before or 2 h after endotoxin challenge significantly suppressed steady-state IL-1β mRNA by 90% and 70%, respectively. LPG also inhibited tumor necrosis factor α and Staphylococcus induction of IL-1β gene expression. The inhibitory effect of LPG is agonist-specific because LPG did not suppress the induction of IL-1β mRNA by phorbol 12-myristate 13-acetate. A unique DNA sequence located within the −310 to −57 nucleotide region of the IL-1β promoter was found to mediate LPG’s inhibitory activity. The requirement for the −310 to −57 promoter gene sequence for LPG’s effect is demonstrated by the abrogation of LPG’s inhibitory activity by truncation or deletion of the −310 to −57 promoter gene sequence. Furthermore, the minimal IL-1β promoter (positions −310 to +15) mediated LPG’s inhibitory activity with dose and kinetic profiles that were similar to LPG’s suppression of steady-state IL-1β mRNA. These findings delineated a promoter gene sequence that responds to LPG to act as a “gene silencer,” a function, to our knowledge, not previously described. LPG’s inhibitory activity for several mediators of inflammation and the persistence of significant inhibitory activity 2 h after endotoxin challenge suggest that LPG has therapeutic potential and may be exploited for therapy of sepsis, acute respiratory distress syndrome, and autoimmune diseases.

Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor.

The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF.

Molecular mechanisms of dexamethasone inhibition of nitric oxide synthase expression in interleukin 1 beta-stimulated mesangial cells: evidence for the involvement of transcriptional and posttranscriptional regulation.

Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) is expressed in rat glomerular mesangial cells upon exposure to the inflammatory cytokine interleukin 1 beta (IL-1 beta). We have reported that nanomolar concentrations of dexamethasone suppress IL-1 beta-induced iNOS protein expression and production of nitrite, the stable end product of NO formation, without affecting IL-1 beta-triggered increase in iNOS mRNA levels. We now have studied the mechanisms by which dexamethasone suppresses IL-1 beta-stimulated iNOS expression in mesangial cells. Surprisingly, nuclear run-on transcription experiments demonstrate that dexamethasone markedly attenuates IL-1 beta-induced iNOS gene transcription. However, this is counteracted by a prolongation of the half-life of iNOS mRNA from 1 h to 2.5 h by dexamethasone. Moreover, dexamethasone drastically reduces the amount of iNOS protein by reduction of iNOS mRNA translation and increased degradation of iNOS protein. These results indicate that glucocorticoids act at multiple levels to regulate iNOS expression, thus providing important insights into the treatment of inflammatory diseases.

Competence for collagenase gene expression by tissue fibroblasts requires activation of an interleukin 1 alpha autocrine loop.

The enzyme collagenase (EC 3.4.24.7), a key mediator in biological remodeling, can be induced in early-passage fibroblasts by a wide variety of agents and conditions. In contrast, at least some primary tissue fibroblasts are incompetent to synthesize collagenase in response to many of these stimulators. In this study, we investigate mechanisms controlling response to two of the conditions in question: (i) trypsin or cytochalasin B, which disrupt actin stress fibers, or (ii) phorbol 12-myristate 13-acetate (PMA), which activates growth factor signaling pathways. We demonstrate that collagenase expression stimulated by trypsin or cytochalasin B is regulated entirely through an autocrine cytokine, interleukin 1 alpha (IL-1 alpha). The IL-1 alpha intermediate also constitutes the major mechanism by which PMA stimulates collagenase expression, although a second signaling pathway(s) contributes to a minor extent. Elevation of the IL-1 alpha level in response to stimulators is found to be sustained by means of an autocrine feedback loop in early-passage fibroblast cultures. In contrast, fibroblasts freshly isolated from the tissue are incompetent to activate and sustain the IL-1 alpha feedback loop, even though they synthesize collagenase in response to exogenous IL-1. We conclude that this is the reason why tissue fibroblasts are limited, in comparison with subcultured fibroblasts, in their capacity to synthesize collagenase. Activation of the IL-1 alpha feedback loop, therefore, seems likely to be an important mechanism by which resident tissue cells adopt the remodeling phenotype.