The polymorphonuclear neutrophils (PMNs) of patients infected with human immunodeficiency virus type 1 (HIV-1) show impaired microbicidal responses. The present study assessed the functional integrity of PMN degranulation responses and the expression of specific receptors that mediate these responses in a group of children vertically infected with HIV-1. PMN degranulation in response to interleukin-8 (IL-8) and complement 5a (C5a) was measured in a group of HIV-1-infected children with mild and severe clinical disease and in an uninfected control group. In addition, the expression of CXCR1, CXCR2, and CD88 on whole-blood PMNs was quantified by flow cytometry. Although CXCR1 expression was found to be largely unaltered in the HIV-1-infected children relative to that in the control children, the intensity of CXCR2 expression was significantly reduced in those with severe disease. Furthermore, there was a significant reduction in the percentage of cells expressing CD88 and in the intensity of CD88 fluorescence in the HIV-1-infected children compared to that in control children, with CD88 fluorescence intensity more significantly reduced in the presence of severe disease. PMNs from a large proportion of the HIV-1-infected children either showed reciprocal degranulation responses or were unresponsive to IL-8 and C5a...
The roles of endogenous cytokines induced by either intact staphylococcal microorganisms or staphylococcal exotoxins were examined using human whole-blood cultures. To accomplish this, interleukin-18 binding protein (IL-18BP) and tumor necrosis factor binding protein (TNFbp) were used to neutralize IL-18 and TNF, respectively, whereas an anti-IL-12 monoclonal antibody was used to neutralize IL-12 and the IL-1 receptor antagonist (IL-1Ra) was used to block IL-1 receptors. Heat-killed Staphylococcus epidermidis and Staphylococcus aureus, as well as the staphylococcal superantigens toxic shock syndrome toxin-1 (TSST-1) and staphylococcus enterotoxin B (SEB) induced gamma interferon (IFN-γ) production. Staphylococcus spp.-induced production of IFN-γ required the presence of endogenous IL-18, IL-12, and TNF. In contrast, TSST-1-induced IFN-γ was not significantly reduced in the presence of IL-18BP, anti-IL-12 antibodies, IL-1Ra, or anti-TNFbp. SEB-induced IFN-γ was significantly inhibited only by anti-IL-12 antibodies, indicating that endogenous IL-18, IL-1, and TNF are not required for SEB-induced IFN-γ. In conclusion, the mechanisms of IFN-γ stimulation by intact staphylococcal microorganisms and by exotoxins differ, and this is likely due to the different receptors which are triggered on the cell membranes. In contrast to its role in the interactions between staphylococci and host cells...
In a previous study, we showed that Staphylococcus aureus supernate (SaS) is a potent agonist for both neutrophils and mononuclear cells. To further investigate the immunomodulating effects of SaS, the effect on different neutrophil receptors was studied. Expression of various neutrophil receptors, before and after treatment with SaS, was quantified by flow cytometry. We found that SaS treatment of neutrophils resulted in a specific and total downregulation of the C5a and the fMLP receptor, both serpentine receptors, while other receptors were totally unaffected. Since these two receptors are both involved in chemotaxis, we tested the effect of SaS in calcium flux and chemotaxis assays. We showed that preincubation with SaS abrogated the rise in intracellular calcium concentration upon triggering with fMLP and C5a. We also showed that SaS is a potent inhibitor of neutrophil chemotaxis towards fMLP and C5a, but does not interfere with chemotaxis towards interleukin-8. These findings indicate that S. aureus produces a virulence factor extracellularly, which impairs chemotaxis towards the infected site.
Initiation of the T-helper lymphocyte activation program is regulated through the T-cell receptor (TCR) and costimulatory receptors. Analysis of TCR and either anti-CD28- or interleukin 1 (IL-1)-mediated activation of the IL-2 promoter shows that costimulatory signals augment promoter activity through NF-κB sites. This study comparatively evaluates the mechanisms whereby signals initiated from the TCR and these two costimulatory receptors converge to synergistically increase NF-κB transcriptional activity. IL-1 alone stimulates an acute but transient NF-κB nuclear localization and a suboptimal NF-κB transcriptional response. In contrast, anti-CD3–anti-CD28 or anti-CD3–IL-1 synergistically stimulate prolonged NF-κB nuclear localization and NF-κB-mediated transcription. Both TCR- and costimulatory receptor-initiated synergistic NF-κB responses result from prolonging high rates of cytosolic IκB degradation during the second phase of the biphasic NF-κB nuclear localization. However, in contrast to previous reports, prolonged nuclear localization of NF-κB complexes is not necessarily associated with long-term depletion of IκBβ. In response to either costimulus, c-Rel selectively translocated to the nucleus as a result of induced c-Rel expression and the continued production of c-Rel–IκBα complexes...
The gastric pathogen Helicobacter pylori is known to activate multiple proinflammatory signaling pathways in epithelial cells. In this study, we addressed the question of whether expression of the interleukin-8 receptors IL-8RA (CXCR1) and IL-8RB (CXCR2) is upregulated in H. pylori-infected human gastric biopsy samples. Biopsy samples from patients infected with H. pylori strains harboring the cag pathogenicity island (PAI) expressed larger amounts of both receptors. In addition, IL-8RB expression was induced in the gastric epithelial cell line AGS upon infection with a clinical isolate containing the cag PAI, while a strain lacking the cag PAI did not. Our finding suggests that gastric epithelial cells express IL-8R in response to H. pylori infection.
Plant disease resistance genes operate at the earliest steps of pathogen perception. The Arabidopsis RPP5 gene specifying resistance to the downy mildew pathogen Peronospora parasitica was positionally cloned. It encodes a protein that possesses a putative nucleotide binding site and leucine-rich repeats, and its product exhibits striking structural similarity to the plant resistance gene products N and L6. Like N and L6, the RPP5 N-terminal domain resembles the cytoplasmic domains of the Drosophila Toll and mammalian interleukin-1 transmembrane receptors. In contrast to N and L6, which produce predicted truncated products by alternative splicing, RPP5 appears to express only a single transcript corresponding to the full-length protein. However, a truncated form structurally similar to those of N and L6 is encoded by one or more other members of the RPP5 gene family that are tightly clustered on chromosome 4. The organization of repeated units within the leucine-rich repeats encoded by the wild-type RPP5 gene and an RPP5 mutant allele provides molecular evidence for the heightened capacity of this domain to evolve novel configurations and potentially new disease resistance specificities.
Interleukin-1 (IL-1) is a key player in inflammation and the immune response. To better understand the complex interactions of IL-1 and its receptors in inflammation, we need to investigate how type I and type II IL-1 receptors (IL-1RI and IL-1RII) are regulated by cytokines and other mediators. Using semiquantitative reverse transcriptase PCR and Northern analysis, we examined the regulation of IL-1RI and IL-1RII mRNA levels in bovine polymorphonuclear leukocytes (PMNs) (i.e., neutrophils) and peripheral blood mononuclear cells (PBMCs) in vitro. IL-1RI mRNA levels were up-regulated in PBMCs by recombinant bovine IL-1beta (rBoIL-1beta), recombinant bovine granulocyte-macrophage colony-stimulating factor (rBoGM-CSF), rBoIL-4, recombinant bovine gamma interferon (rBoIFN-gamma), and dexamethasone. IL-1RI mRNA was increased in bovine PMNs exposed to rBoGM-CSF, rBoIL-4, and dexamethasone but was down-regulated by rBoIL-1beta and rBoIFN-gamma. IL-1RII mRNA was increased in bovine PBMCs and PMNs after exposure to rBoIL-1beta, rBoGM-CSF, rBoIL-4, and dexamethasone. In contrast, rBoIFN-gamma down-regulated the expression of bovine IL-1RII mRNA in PBMCs. These findings suggest that the expression of bovine IL-1RI and IL-1RII mRNAs is regulated differently by certain soluble stimuli (e.g....
This study was undertaken to determine the effect of Pseudomonas aeruginosa alkaline protease (AP) and elastase (ELA) on human lymphocyte function. AP at 50 micrograms/ml and ELA at 12 micrograms/ml caused a 50% inhibition of phytohemagglutinin-induced proliferation. There was no difference in the effect of proteases on CD4- and CD8-positive cells. To determine the effect of proteases on interleukin-2 (IL-2)-induced cell proliferation, the proteases and IL-2 were added to the IL-2-dependent CTLL-2 cell line. AP and ELA inhibited the proliferation of these cells. When IL-2 was added in excess, the inhibition was partly reversed. ELA at 10 micrograms/ml cleaved IL-2, as judged by size chromatography of a reaction mixture containing 125I-labeled IL-2 and the proteases. The ELA-digested IL-2 exhibited a reduced binding capacity to IL-2 receptors on the lymphocytes. Furthermore, treatment of phytohemagglutinin-stimulated lymphocytes with AP and ELA resulted in inhibition of binding of intact IL-2 to IL-2 receptors on the stimulated lymphocytes. These results indicated that P. aeruginosa-derived enzymes are able to interfere with human lymphocyte function in vitro and that this effect might be due to cleavage of IL-2.
Increased levels of soluble interleukin-2 receptors (IL-2R) in serum were observed in both Plasmodium falciparum- and P. vivax-infected individuals compared with nonparasitemic subjects. Clinical symptoms of P. falciparum malaria were associated with higher levels of soluble IL-2R. Temporal evolution in serum of IL-2R during the course of a malaria attack mimicked the kinetics of soluble IL-2R under experimental conditions.
Cell surface components of viridans streptococci and enterococci have been shown to stimulate the release of tumor necrosis factor alpha (TNF) and interleukin-6 from monocytes/macrophages. In the sera from 10 patients with subacute enterococcal or streptococcal endocarditis, however, the levels of both cytokines were low or undetectable, with elevated TNF levels on admission in 3 patients with complicated disease. Soluble TNF receptor levels were significantly elevated compared with those of healthy controls. When patients with malaria were used as a control group of acute intravascular infection with high circulating TNF values, the ratio between soluble TNF receptors and TNF on admission was significantly greater in the patients with subacute bacterial endocarditis. Besides different amounts of circulating TNF, enhanced TNF receptor shedding may have an important role in the pathogenesis of subacute versus acute clinical disease following human intravascular infection.
The Tac protein plays a role in high- and low-affinity interleukin 2 (IL-2) receptors. A mutational survey of this molecule identified several small segments in which the binding of IL-2 was particularly sensitive to amino acid substitutions. Two of the segments (residues 1-6 and 35-43) located in the exon 2-encoded region of the molecule overlapped the apparent binding sites of three monoclonal antibodies (anti-Tac, GL439, and H31) that block high- and low-affinity Tac-IL-2 interactions, thus supporting the hypothesis that these segments of the protein are at or near sites of receptor-ligand contact. In contrast, the apparent binding sites of antibodies (Hiei and H47) that selectively inhibit high-affinity IL-2 binding were mapped to a distinct location (residues 158-160) within the region encoded by exon 4 of the Tac gene. Since high-affinity receptors consist of a heterodimer of Tac and a second ligand-binding protein (p70), this portion of the Tac molecule may be involved in the interaction between the two receptor subunits. As expected, the binding sites of noninhibitory antibodies (7G7/B6, residues 140-144; H48, residues 170-211) did not overlap those segments in which IL-2-binding mutants were observed. These results provide a preliminary correlation of structure and function for the Tac protein that should prove useful in evaluating detailed models of the IL-2-receptor complex.
We demonstrated that interleukin 1 (IL-1), a potent peptide mediator in immune and inflammatory responses, stimulates the synthesis of cAMP in a variety of IL-1-responsive cell targets. We also showed that cAMP analogs and cAMP-inducing agents can replace IL-1 in the induction of interleukin 2 receptors on lymphocytes as well as in phytohemagglutinin-induced murine thymocyte proliferation. By use of IL-1 and the cAMP-inducer, forskolin, a direct correlation between the induced level of cAMP and the degree of lymphocyte interleukin 2 receptor expression or thymocyte proliferation was established. Our results indicate that cAMP may be an important intracellular second messenger for IL-1.
A chimeric toxin composed of human interleukin 6 (IL-6) attached to a portion of Pseudomonas exotoxin (PE) devoid of its own cell recognition domain has been produced in Escherichia coli. The fusion protein (IL-6-PE40) is cytotoxic to a human myeloma cell line expressing IL-6 receptors but has no effect on IL-6 receptor-negative cells. The specificity of IL-6-PE40 cytotoxicity was demonstrated through competition with excess IL-6 and neutralization with an antibody to IL-6. IL-6-PE40 may be useful in the selective elimination of myeloma cells and other cells with high numbers of IL-6 receptors.
125I-labeled recombinant human interleukin 3 (IL-3) bound, at 4 degrees C, to a single class of high-affinity receptors on human eosinophils with an apparent dissociation constant (Kd) of 470 pM, but it did not bind to human neutrophils. 125I-labeled recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) also bound to a single class of high-affinity receptors on eosinophils with an apparent Kd of 44 pM and on neutrophils with an apparent Kd of 70 pM. These binding characteristics were consistent with the biological activities of IL-3 and GM-CSF on eosinophils and with the lack of stimulation of neutrophil function by IL-3. Specificity studies under conditions shown to prevent receptor internalization showed that the binding of 125I-labeled IL-3 to eosinophils was partially inhibited by GM-CSF but not by other cytokines. Reciprocal experiments with 125I-labeled GM-CSF showed that IL-3 but not other cytokines partially inhibited binding to eosinophils. In contrast, the binding of 125I-labeled GM-CSF to neutrophils was not inhibited by IL-3 or other cytokines tested. Quantitative inhibition binding experiments on eosinophils showed that the reciprocal inhibition between IL-3 and GM-CSF was not complete up to a concentration of heterologous ligand of 100 nM. These results show that (i) IL-3 binds to eosinophils but not neutrophils and (ii) IL-3 and GM-CSF specifically interact on the surface of eosinophils...
We have investigated whether interleukin 3 (IL-3) supports the growth of cells of different lineages by the same mechanism(s). The experiments were carried out with Ea3 cells, a mouse pre-B cell line, and S-480-3 cells, a mouse basophil cell line, both of which are totally IL-3 dependent. We found that Ea3 lymphocytes but not S-480-3 basophils absorb partially purified IL-3. Both Ea3 and S-480-3 cells respond to IL-3 by increasing anaerobic glycolysis as determined by lactic acid production. S-480-3 cells responded to exogenous ATP by maintaining proliferation and reducing lactic acid production, but Ea3 lymphocytes are refractory to exogenous ATP. We conclude that there may be two distinct mechanisms by which cells respond to IL-3, indicated by early events concerning the binding of IL-3 and the effect of exogenous ATP on respiratory metabolism. One appears to be a ligand-receptor-mediated mechanism in lymphoid cells and the other to be a mechanism that is partially replaceable by exogenous ATP in nonlymphoid cells not associated with lymphoid-like receptors. Our findings may explain (i) the apparent variety of cell lineages promoted by IL-3 by a widely available mechanism that supports glycolysis and, therefore, enables both proliferation and possibly expression of binding sites for lineage specific differentiation factors and (ii) the existence of lymphocytes that express receptors specific for IL-3 and are inducible for other characteristics and functions in a regulated manner.
Human T-cell clones and anti-T-cell-receptor antibodies (clonotypic) directed at surface receptors for antigen (T3-Ti molecular complex) as well as anti-interleukin 2 (IL-2) and anti-IL-2-receptor antibodies were utilized to investigate the mechanism by which alloantigens or antigen plus self-major histocompatibility complex (MHC) (i.e., physiologic ligand) trigger specific clonal proliferation. Soluble or Sepharose-bound anti-Ti monoclonal antibodies, like physiologic ligand, enhanced proliferative responses to purified IL-2 by inducing a 6-fold increase in surface IL-2 receptor expression. In contrast, only Sepharose-bound anti-Ti or physiologic ligand triggered endogenous clonal IL-2 production and resulted in subsequent proliferation. The latter was blocked by antibodies directed at either the IL-2 receptor or IL-2 itself. These results suggest that induction of IL-2 receptor expression but not IL-2 release occurs in the absence of T3-Ti receptor cross-linking. Perhaps more importantly, the findings demonstrate that antigen-induced proliferation is mediated through an autocrine pathway involving endogenous IL-2 production, release, and subsequent binding to IL-2 receptors.
Members of the newly identified receptor family for cytokines characteristically lack the intrinsic protein tyrosine kinase domain that is a hallmark of other growth factor receptors. Instead, accumulating evidence suggests that these receptors utilize nonreceptor-type protein tyrosine kinases for downstream signal transduction by cytokines. We have shown previously that the interleukin-2 receptor beta-chain interacts both physically and functionally with a Src family member, p56lck, and that p56lck activation leads to induction of the c-fos gene. However, the mechanism linking p56lck activation with c-fos induction remains unelucidated. In the present study, we systematically examined the extent of c-fos promoter activation by expression of a series of p56lck mutants, using a transient cotransfection assay. The results define a set of the essential amino acid residues that regulate p56lck induction of the c-fos promoter. We also provide evidence that the serum-responsive element and sis-inducible element are both targets through which p56lck controls c-fos gene activation.
Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.
The normal cellular counterpart of the v-fms oncogene product is a receptor for the mononuclear phagocyte colony-stimulating factor, CSF-1. An interleukin-3 (IL-3)-dependent mouse myeloid cell line, FDC-P1, was infected with a murine retrovirus vector containing v-fms linked to a gene encoding resistance to neomycin (neo). Infected cells selected for resistance to the aminoglycoside G418 contained few proviral DNA copies per haploid genome, expressed low levels of the v-fms-coded glycoprotein, remained IL-3 dependent for growth, and were nontumorigenic in nude mice. In contrast, infected cells selected for their ability to grow in the absence of IL-3 contained an increased number of proviral insertions, expressed high levels of the v-fms-coded glycoprotein, and were tumorigenic in nude mice. The IL-3-independent cells expressed IL-3 receptors of comparable number and affinity to those detected in uninfected FDC-P1 cells and did not produce a growth factor able to support replication of the parental cells. Thus, the synthesis of high levels of the v-fms gene product in FDC-P1 cells abrogated their requirement for IL-3 and rendered the cells tumorigenic by a nonautocrine mechanism. The data suggest that v-fms encodes a promiscuous tyrosine kinase able to transform cells of the myeloid lineage that do not normally express CSF-1 receptors.
Tac antigen, the receptor for human interleukin 2 (IL-2), contains in its intracytoplasmic region a serine residue (Ser-247) that is seemingly the predominant site of protein kinase C-mediated phosphorylation. A number of studies on growth factor receptors have suggested the importance of phosphorylation in receptor structure, function, and regulation. In this study, we generated site-directed mutations in the Tac antigen cDNA to generate mutant receptors in which Ser-247 or Thr-250, a probable site of minor phosphorylation, was replaced with another amino acid that is not accessible to phosphorylation. Study of the expression of these mutant genes in a T-lymphoid cell line has provided no evidence as to the essential role of the above-mentioned residues in determining the degree of receptor affinity, its ability for signal transduction, and phorbol ester-mediated regulation of the receptor. Our results strongly suggest the existence of an IL-2 receptor "complex" in which the Tac antigen is associated with another molecule(s) that is involved in receptor structure, function, and regulation.