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Human gammadelta T lymphocytes respond to viral, bacterial, protozoal, and tumoral antigens, but their precise function remains unknown. In adults the major circulating gammadelta T-cell subset expresses the Vgamma9Vdelta2 T-cell receptor and responds to protease-resistant phosphorylated derivatives found in many pathogens. In this study we show that activation of Vdelta2(+) cells with the nonpeptidic antigen isopentenyl pyrophosphate (IPP) rapidly induces (within 4-12 hours) the C-C chemokines MIP-1alpha, MIP-1beta, and lymphotactin but not MCP-1. The most robust response was obtained for MIP-1beta. IPP induction of MIP-1alpha and MIP-1beta was not affected by costimulation with interleukin-4 (IL-4), IL-10, TGF-beta, or interferon-gamma (INF-gamma). However, IL-12 significantly enhanced IPP-induced expression and release of MIP-1alpha that was down-regulated by TGF-beta whereas the induction of MIP-1beta by IPP+IL-12 was refractory to cotreatment with TGFbeta indicating that these chemokines are differentially regulated by these cytokines. Vdelta2(+) T cells also expressed a wide range of C-C chemokine receptors including CCR1, CCR5, and CCR8, all of which were down-regulated following activation. We conclude that Vdelta2(+) cells can be rapidly induced by components of bacterial cell walls to express high levels of proinflammatory chemokines, supporting an important role for these cells in the early stages of the inflammatory responses to many common pathogens. (Blood. 2000, 95:39-47)

Interleukin-1 receptor antagonist (IL-1Ra) is produced by hepatocytes with characteristics of an acute-phase protein. To examine the role of IL-4 and IL-13 in production of IL-1Ra, human primary hepatocytes and HepG2 human hepatoma cells were cultured in the presence of IL-4 or IL-13 in combination with IL-1beta and/or IL-6. The results indicated that both IL-4 and IL-13 amplified the stimulatory effect of IL-1beta on production of IL-1Ra protein and messenger RNA (mRNA) by both human primary hepatocytes and HepG2 cells. IL-1Ra refers to three different peptides, one secreted (sIL-1Ra) and two intracellular (icIL-1RaI and icIL-1RaII), derived from the same gene. sIL-1Ra and icIL-1RaI are the products of two different mRNA, whereas icIL-1RaII is synthesized by alternative translation initiation mainly from sIL-1Ra mRNA. Our results show that both sIL-1Ra and icIL-1RaII, but not icIL-1RaI, are produced by HepG2 cells and human hepatocytes. Transient transfection experiments as well as mRNA stability studies indicated that IL-4 stimulated sIL-1Ra production primarly at the level of transcription. Gel retardation assays showed that IL-4 induced the formation of a STAT6-DNA complex with a STAT6 binding element within the sIL-1Ra promoter, but had no effect on IL-1-induced NF-kappaB binding activity. In contrast to IL-1Ra, production of C-reactive protein by human primary hepatocytes was stimulated by IL-6 and decreased by the addition of IL-4.

Interleukin-1beta (IL-1beta) is considered an important mediator in the pathogenesis of septic shock or bacterial meningitis. Its activity is specifically modulated by IL-1 receptor antagonist (IL-1Ra) and IL-1 soluble receptor type II (IL-1sRII). We now describe the time-course of IL-1beta and these modulating agents in 59 patients with acute meningococcal infections, the prototype human disease of acute endotoxin exposure. Plasma IL-1beta was increased only in severe shock and normalized within 12 to 24 hours, indicating that patients were admitted in an early stage of cytokine activation. Increased IL-1beta values in cerebrospinal fluid (CSF) were confined to patients with meningitis. Plasma IL-1Ra was elevated in both shock and nonshock patients, extremely high values being measured in severe shock. High concentrations of IL-1Ra in CSF were found in meningitis. Plasma IL-1Ra peaked shortly after IL-1beta and decreased steeply in 1 to 2 days, followed by sustained moderately elevated levels in shock patients. Interestingly, IL-1sRII showed a completely different pattern. At admission, both nonshock and shock patients manifested a similar moderate increase of plasma IL-1sRII. However, during recovery plasma IL-1sRII further increased reaching maximal concentrations 3 to 5 days after admission, 1 to 2 days after normalization of IL-1Ra. In shock patients this increase was more prominent than in nonshock patients. It is hypothesized that this increase in plasma IL-1sRII can be explained by a synergistic effect of dexamethasone and endotoxin. A second interesting observation was that, unlike the pattern in plasma, IL-1sRII levels in CSF paralleled those of IL-1beta and IL-1Ra. This suggests different modulation of IL-1beta activity in the subarachnoid space and the plasma compartment. We conclude that: (1) During the early stage of meningococcal infections IL-1Ra modulates IL-1 activity, whereas during recovery IL-1sRII may be more important. (2) Modulation in CSF and in the plasma compartment are differentially regulated.

Effects of soluble recombinant human type I interleukin-1 receptor (sIL-1RI) were evaluated in 18 volunteers given intravenous endotoxin and randomized to placebo (n = 6), low-dose (n = 6), or high-dose (n = 6) sIL-1RI. Soluble IL-1RI decreased IL-1 beta (P = .001), but decreased IL-1ra (P = .0001), and resulted in 10-fold and 43-fold dose-related increases in sIL-1RI-IL-1ra complexes compared with placebo (P < or = .001). High-dose sIL-1RI was associated with increased levels of immunoactive tumor necrosis factor-alpha (P = .02), IL-8 (P = .0001), and cell-associated IL-1 beta (P = .047). C-reactive protein levels were higher after sIL-1RI than placebo (P = .035). Soluble IL-1RI decreased the severity of chills (P = .03), but did not alter other symptoms, changes in temperature, systemic hemodynamic responses, or changes in leukocyte and platelet number. Thus, sIL-1RI had no discernable antiinflammatory effect following endotoxin administration due in part to low levels of circulating IL-1 beta and neutralization of IL-1ra inhibitory function. This latter interaction represents an indirect mechanism of agonist activity elicited by sIL-1RI and may contribute to increases in inflammatory mediators, limiting therapy with sIL-1RI during endotoxemia.

Specific glycoforms of CD43, the major O-glycosylated cell-surface protein on T lymphocytes, can affect cell adhesion according to the types of carbohydrate side chains carried. In the peripheral immune system, CD43 130 kD, which carries core 2 O-glycan structures on its surface, is an activation antigen expressed on both CD4 and CD8 single-positive (SP) T cells. We have previously shown that the 115-kD resting and 130-kD activation glycoforms of murine CD43 are differentially regulated on peripheral SP T cells. In this study, we used transgenic mice expressing T-cell receptors (TCRs) specific for antigens presented by class I and class II major histocompatibility complex (MHC) molecules to determine whether CD43 glycoforms are involved in thymocyte differentiation. Positive selection in these mice results in an increase in the production of CD8 and CD4 SP T cells, respectively, which express the transgenic TCR. Positive selection is also accompanied by the upregulation of TCR, CD69, and CD5. Using these markers to define stages of thymocyte maturation, we found that CD43 130 kD was downregulated in the positive selection of CD4 CD8 double-positive thymocytes expressing a class I but not class II MHC-restricted TCR. These data suggest that core 2 glycosyltransferase (C2GnT) modulated expression of CD43 glycoforms may be involved in thymic selection events.

Interleukin-13 (IL-13) belongs to the IL-4 gene family. Like IL-4, IL-13 induces IL-1 receptor antagonist (IL-1Ra) synthesis with no effect on IL-1beta synthesis. We investigated whether IL-13 induces IL-1Ra synthesis via a pathway similar to IL-4. In human peripheral blood mononuclear cells, IL-13 (1 to 100 ng/mL alone induced IL-1Ra synthesis in a dose-dependent manner. A single amino acid mutant form of IL-4 (hIL4.Yl24D) induced IL-1Ra synthesis, acting as a partial agonist. However, hIL-4.Yl24D inhibited IL-1Ra synthesis induced by either IL-4 or IL-13. IL-13 alone induced accumulation of IL-1Ra mRNA. Furthermore, IL-13 reduced steady- state levels for IL-1beta mRNA but enhanced those for IL-1Ra mRNA in cells stimulated with lipopolysaccharide (LPS) or IL-1alpha. Accordingly, IL-13 suppressed IL-1beta synthesis but enhanced IL-1Ra synthesis in these cells. IL-13 reduced the stability of IL-1beta mRNA (2.9 v 1.7 hours) but failed to modify the stability of IL-1Ra mRNA (2.7 v 2.5 hours). Moreover, IL-13 induced transcriptional activation of the IL-1Ra gene, but reduced IL-1beta gene transcription. Our results suggest that the commonality between IL-13 and IL-4 in inducing IL-1Ra synthesis results from the engagement of a subunit common to both receptors.

Neutrophils and macrophages represent the first line of defense against microbial invaders. However, the role of phagocytes in host response to viral infection is poorly understood. We have previously shown that Epstein-Barr virus (EBV) interacts with human monocytes and modulates cytokine production in this cell type, but its effects on neutrophils are still unknown. In the present study, we investigated the presence of EBV receptor (CR2 or CD21) on neutrophils by cytofluorometry using five different anti-CD21 monoclonal antibodies (MoAbs), as well as fluoroscein isothiocyanate-EBV (FITC-EBV). Whereas no significant amount of neutrophils reacted with anti-CD21 MoAbs, studies with FITC-EBV indicated that viral particles bind to 30% of cells (in some individuals, EBV binds to more than 50% of neutrophils). This interaction is specific as it was completely inhibited by nonconjugated virus or with labeled virus preincubated with neutralizing MoAbs. After EBV treatment, cellular aggregation was observed in neutrophil cultures, an indication that neutrophils were activated. Although EBV did not induce respiratory burst activity in neutrophils, pretreatment with infectious particles enhanced (priming effect) the fMLP-induced O2- release in neutrophils. Instead of restricting our analysis to specific cytokine genes, we investigated the effects of EBV on neutrophil transcriptional events in general. The effect of this virus on de novo synthesis of total cellular RNA was first investigated by measuring the incorporation of [5-3H] uridine into total RNA. The results showed that RNA synthesis in neutrophils was significantly increased (2.3- to 21.3-fold) by EBV compared with the unstimulated controls. Live and UV-inactivated virus markedly induced RNA synthesis, whereas heat-inactivated virus lost this ability. Induction of RNA transcription was EBV specific, as an EBV-neutralizing antiserum abolished this effect. Induction of protein synthesis was also studied by measuring the incorporation of [35S] methionine and [35S] cysteine into secreted and intracellular proteins in neutrophils incubated with EBV. The synthesis of both secreted and cytoplasmic proteins was induced by EBV. One- and two-dimensional gel electrophoresis analysis showed that EBV modulates protein synthesis, because activation of the synthesis of certain proteins was accompanied by the inhibition of others. Interleukin-1 beta (IL-1 beta) and IL-1 receptor antagonist (IL-1Ra) synthesis was found to be induced by EBV. Therefore, modulation of host-response proteins such as IL-1Ra could be one of the many mechanisms by which this virus avoids rejection.(ABSTRACT TRUNCATED AT 400 WORDS)

CD43, a prevalent white blood cell molecule distinguished by its mucin-like surface region, has been proposed as a "functional barrier" that prevents or negatively regulates a variety of cell surface interactions. Implicit in this hypothesis is the expectation that CD43 will be altered or removed when white blood cells are activated. To investigate alterations of CD43 in a dramatic example of functional cell activation, suspension neutrophils were challenged with opsonized zymosan, a characterized stimulator of phagocytosis and respiratory burst oxidase. Flow cytometry showed decreased surface density of CD43 in opsonized zymosan-treated neutrophils, and immune precipitation showed decreased cellular CD43 content, indicating that opsonized zymosan downregulates CD43 by a proteolytic mechanism. Based on densitometry of immune precipitates, CD43 levels were decreased 42% +/- 6% in neutrophils treated for 10 minutes with opsonized zymosan and decreased 70% +/- 3% in neutrophils treated with phorbol 12-myristate 13-acetate (PMA). CD43 downregulation in response to opsonized zymosan, like PMA-induced CD43 downregulation, was insensitive to the serine protease inhibitor diisopropylfluorophosphate (DFP). In contrast, CD43 downregulation in response to opsonized zymosan or PMA was prevented by 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF) and 3'4'-dichloroisocoumarin (3,4-DCI), both of which are characterized serine protease inhibitors. Activation of the neutrophil respiratory burst oxidase by opsonized zymosan or PMA was also insensitive to DFP and prevented by AEBSF and 3,4-DCI. These findings indicate a requirement for a proteolytic step in activation of the respiratory burst of intact suspension neutrophils by opsonized zymosan and PMA and suggest that CD43 cleavage may be a required proteolytic event.

We investigated the effects of interferon-alpha (IFN-alpha) on the expression of cytokines by human bone marrow stromal cells. Production of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-CSF (G-CSF), and interleukin-1 beta (IL-1 beta) in stromal cell layers was induced by incubation with IL-1 alpha, tumor necrosis factor (TNF), or lipopolysaccharide (LPS). Addition of IFN-alpha to such stimulated cultures resulted in a strong downregulation of mRNA expression of GM-CSF and IL-1 beta. Similarly, the protein levels of GM-CSF and IL-1 beta were significantly reduced by IFN-alpha, whereas G-CSF production was only moderately inhibited. In contrast, IFN-alpha markedly stimulated the production of IL-1 receptor antagonist (IL-1RA) by stromal cells. The inhibition of cytokine expression resulted in a reduced hematopoietic activity of stromal cells, indicated by a reduced proliferation of the factor dependent cell line MO7e on IFN-alpha-treated stromal cells. In the presence of cycloheximide (CHX), IFN-alpha failed to inhibit IL-1 mRNA expression, whereas the regulation of GM-CSF and IL-1RA by IFN-alpha was not affected. Our results indicate that the myelosuppressive effects of IFN-alpha, as observed in therapeutic applications or associated with viral infections, are, in part, indirectly mediated by inhibition of the paracrine production of hematopoietic growth factors.

The aim of this study was to examine the expression of interleukin-1 receptor antagonist (IL-1ra) in human myelomonocytic cells treated with IL-13. IL-13 induced IL-1ra transcripts in human circulating monocytes and polymorphonuclear cells (PMN). Induction by IL-13 was not blocked, but rather superinduced, in the presence of the protein synthesis inhibitor cycloheximide. Actinomycin D blocked induction, suggesting involvement of gene transcription. The half-life of IL-1ra transcripts was prolonged by IL-13 from 1.3 hours to 4.5 hours in monocytes and to 12 hours in PMN. By reverse transcriptase-polymerase chain reaction, IL-13 was found to augment the transcripts coding for the soluble form of IL-1ra, but also to induce the expression of the intracellular (keratinocyte) form of IL-1ra, the latter being extremely low or undetectable in myelomonocytic cells. IL-13 induced production of IL-1ra in myelomonocytic cells, augmenting both cell-associated and released protein. Induction of IL-1ra by IL-13 may represent a further mechanism by which this molecule can counteract the potent proinflammatory properties of IL-1.

The aim of this study was to investigate whether interleukin (IL)-6 induces the production of IL-1 and tumor necrosis factor (TNF) antagonists. Serial plasma samples were obtained from cancer patients participating in phase I and II trials of recombinant IL-6 administered as a 120-hour continuous intravenous (IV) infusion. Plasma IL-1 receptor antagonist (IL-1Ra) and soluble TNF receptor p55 (TNFsRp55) levels were measured by specific radioimmunoassays (RIAs). IL-1Ra levels increased rapidly, reaching peak values (9.6 +/- 1.7 ng/mL) within 2 to 4 hours of beginning treatment. Thereafter, levels promptly declined, reaching near baseline within 24 hours despite continuation of IL-6. TNFsRp55 plasma levels increased within 4 to 8 hours after initiating treatment and increased progressively throughout the duration of therapy. IL-1 beta and TNF-alpha plasma levels were below the detection limit in all samples tested. Peripheral blood mononuclear cells (PBMC) exposed to IL-6 produced only small amounts (1.56 +/- 0.3 ng/mL) of IL-1Ra, even in the presence of exogenous soluble IL-6 receptor (gp80). TNFsRp55 levels measured in the supernatants of IL-6-stimulated PBMC were below the detection limit of the assay. Macrophages generated by culturing monocytes in granulocyte-macrophage colony-stimulating factor (GM-CSF) were much more responsive to IL-6 than freshly isolated unfractionated or adherent PBMC and synthesized almost as much IL-1Ra when stimulated with IL-6 as with endotoxin. These results suggest that the antinflammatory properties of IL-6 may be due; in part, to the induction of IL-1Ra synthesis and the release of soluble TNF receptors. Our findings also suggest that tissue macrophages may be an important source of IL-6-induced IL-1Ra.

The beta-glucan receptor, found on monocytes and neutrophils, binds glucose polymers derived from fungi. Ligands for the receptor have various immunomodulatory effects, including increased microbicidal killing activity. We have investigated the effect of beta-glucans on the production of interleukin-1 (IL-1) and its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1Ra). Particulate beta-glucan induced IL-1Ra production from human peripheral blood mononuclear cells (PBMC) but did not stimulate IL-1 beta synthesis or gene expression in these same cells. Monomeric (soluble) beta-glucan did not induce IL-1Ra production. However, when preincubated with PBMC, monomeric beta-glucan significantly (P < .01) reduced particulate beta-glucan induction of IL-1Ra by 40%, suggesting that crosslinking of beta-glucan receptors is required for induction of IL-1Ra. In support of this, monomeric beta-glucan immobilized on plastic surfaces stimulated IL-1Ra production. Vitamin D3, which increases the functional capacity of beta-glucan receptors, increased IL-1Ra production induced by particulate beta-glucan, whereas dexamethasone suppressed IL-1Ra synthesis. Because of their differential effects on cytokine production, beta-glucans may be used to therapeutic advantage in the diseases in which IL-1 is implicated.

Leukocytes express a major sialoglycoprotein, leukosialin, of which the apparent molecular weight (mol wt) can be variable according to the differences in O-glycans attached to this molecule. In the present study, we analyzed the structures of O-glycans attached to leukosialin present in various T-lymphocytic leukemia cells. T-lymphoid cells from patients with acute T-lymphocytic leukemia express a large amount of the branched hexasaccharides, NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc, which are also expressed in activated normal T lymphocytes, but that are almost absent in resting normal T lymphocytes. T-lymphoid cells from patients with chronic T-lymphocytic leukemia, on the other hand, mainly express the tetrasaccharides NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAc on leukosialin, but they also express a small significant amount of the hexasaccharides. The same hexasaccharides can be detected in thymocytes. The increased amount of the hexasaccharides in acute leukemia is associated with increased activity of beta 1----6 GlcNAc-transferase, a key enzyme in forming the hexasaccharides. Immunoblot analysis of cell lysates showed that monoclonal antibody (MoAb) T-305 reacts preferentially with leukosialin of high mol wt containing the hexasaccharides. These findings indicate that T-lymphocytic leukemia cells reexpress the oligosaccharides present in immature cells.

The major sialoglycoprotein of human erythrocyte membranes (glycophorin) is one of the most-studied membrane proteins. Although the structure is relatively well known, almost nothing is known about its expression in erythroid cells. To study this we raised an antiserum that reacted specifically with this protein. This was accomplished by immunization of rabbits with a preparation of glycophorin followed by absorption with En(a-) erythrocyte membranes, which lack glycophorin. By use of this antiserum and a staphylococcus protein A technique we could establish that only bone marrow cells of erythrocyte lineage express glycophorin at the cell surface. This occurs in basophilic normoblasts and later stages of erythrocyte differentiation, whereas pronormoblasts do not seem to contain glycophorin.