IL-1 alpha or tumor necrosis factor-alpha stimulate release of three NAP-1/IL-8-related neutrophil chemotactic proteins in human dermal fibroblasts

Human dermal fibroblasts in culture secrete three protein-like neutrophil chemotactic factors, when stimulated either with human rIL-1 alpha or IL-1 beta; not, however, after incubation with LPS. These three fibroblast-derived neutrophil-activating proteins (FINAP) could be purified by subsequently performed reversed phase and size exclusion HPLC. By high resolution SDS-PAGE, all the proteins were shown to migrate with an Mr of 6,700 (alpha-FINAP), 3,600 (beta-FINAP), and 5,300 (gamma-FINAP). All purified cytokine preparations were found to be chemotactic for human neutrophils. In addition, all FINAP induced release of lysosomal enzymes in neutrophils. Deactivation of chemotaxin-elicitable enzyme release showed cross-desensitization of all FINAP with NAP-1/IL-8. Western blot analysis of alpha-FINAP by using mAb against neutrophil-activating protein (NAP)-1/IL-8 reveals immunologic cross-reactivity with NAP-1/IL-8. By amino-terminal amino acid sequence analysis alpha-FINAP could be identified as the 77-residue extended form of NAP-1/IL-8 containing the 79-residue form as a minor contaminant. Whereas beta-FINAP has been found to be a truncation product of alpha-FINAP, gamma-FINAP shows identity with authentic melanoma growth stimulatory activity with respect to retention time upon reversed phase HPLC, high resolution SDS-PAGE, and biologic properties, as well as amino-terminal amino acid sequence. These data show that human dermal fibroblasts may actively participate in inflammatory reactions by secretion of proinflammatory cytokines.     

NAP-1/IL-8 induces up-regulation of CR1 receptors in human neutrophil leukocytes

The effect of the neutrophil-activating peptide NAP-1/IL-8 on the expression of complement receptor type 1 (CR1) in human neutrophils was studied. NAP-1/IL-8 enhanced CR1 expression at concentrations between 10(-10) and 10(-8) M. The maximum increase with respect to unstimulated control cells was on average 2.3 fold. The effect was rapid: Half-maximum enhancement was obtained in 4 min and the plateau was reached in 15 min. The chemotactic peptide fMLP, tested for comparison, was effective between 10(-9) and 10(-7) M, showed a similar time course and a somewhat higher maximum effect (2.8 fold increase). The effect of NAP-1/IL-8 was prevented by pretreatment of the cells with B.pertussis toxin and desensitization was observed following restimulation. Stimulus combination experiments suggested that NAP-1/IL-8 mobilizes the same or a similar intracellular pool of CR1 receptors as fMLP or C5a.     

Interleukin-8 has antitumor effects in the rat which are not associated with polymorphonuclear leukocyte cytotoxicity

The antitumor effect of lipopolysaccharides (LPS) has been observed in several experimental models and is likely to be mediated by macrophages. Stimulation of macrophages with LPS results in the release of several cytokines, including tumor necrosis factor, interleukin-1 and neutrophil-activating peptide-1/interleukin-8 (IL-8), which activates polymorphonuclear leukocytes (PMN) in vitro. Since PMN have an antitumor activity, we tested the in vivo effect of IL-8 on the growth of peritoneal carcinomatoses induced by PROb colon cancer cells in syngeneic rat. IL-8 induced a significant regression of tumors measuring 1–5 mm, and a complete regression was observed in 8 out of 40 rats in four independent experiments. IL-8 was not directly cytotoxic in vitro for tumor cells and was effective in vivo in a narrow range of doses. IL-8 had a significant chemotactic effect for peritoneal PMN in both normal and tumor-bearing rats. PMN taken from the peritoneum of tumor-bearing rats during IL-8 treatment had the same cytotoxic activity against PROb tumor cells as PMN from untreated control rats. Microscopic examinations of tumors during the treatment showed poor infiltrating by PMN. We conclude that the antitumor activity of IL-8 in this model is not mediated by PMN cytotoxicity.     

Selected antibodies to leukocyte common antigen (CD45) inhibit human neutrophil chemotaxis.

The CD45 Ag family is a group of high m.w. glycoproteins that are expressed on the plasma membranes of all leukocytes. CD45 has protein tyrosine phosphatase activity and appears to regulate signal transduction and lymphocyte activation by specific association with receptor molecules on T and B lymphocytes. However, little is known about CD45 function in neutrophils (PMN). In this study, PMN were incubated with CD45 mAb and tested for their chemotactic responses to four unrelated chemo-attractants: FMLP, leukotriene B4 (LTB4), recombinant human C5a (C5a), and recombinant human neutrophil-activating protein-1, recently designated IL-8. A panel of CD45 mAb including an IgM mAb, AHN-12.1, and six IgG1 mAb, AHN-12, AHN-12.2, AHN-12.3, AHN-12.4, HLe-1, and KC56(T200), were tested for their effects on PMN chemotaxis. PMN chemotaxis was evaluated with two different membrane assays; one assay quantified the total number of migrating PMN and the other assayed the leading front of migrating PMN. AHN-12.1 and KC56(T200) significantly inhibited PMN chemotaxis to LTB4 and C5a. AHN-12.1 slightly inhibited PMN chemotaxis to FMLP, but KC56(T200) did not. In contrast, AHN-12 and HLe-1 did not significantly inhibit PMN chemotaxis to any of the chemoattractants. None of the CD45 mAb inhibited PMN chemotaxis to neutrophil-activating protein-1/IL-8. None of the CD45 mAb inhibited PMN superoxide production. These results suggest that PMN CD45 epitopes may interact with LTB4 and C5a receptor-associated molecules and regulate chemotactic responses.     

Purification and partial biologic characterization of a human lymphocyte-derived peptide with potent neutrophil-stimulating activity

A novel neutrophil-activating peptide is detected in supernatants from mitogen-stimulated human T lymphocyte preparations. This chemotaxin was purified to apparent homogeneity by sequential Gel G-75 permeation chromatography, wide pore reversed phase (RP-8) HPLC, size exclusion HPLC, and reversed phase (RP-18) HPLC. Additional characterization of this lymphocyte-derived neutrophil-activating peptide (LYNAP) resulted in a single peak upon reversed phase HPLC and size exclusion HPLC. SDS-PAGE under nonreducing conditions revealed a single line at 10 kDa. LYNAP stimulated neutrophil chemotaxis (ED50 of 3 +/- 3 ng/ml), chemokinesis (ED50 of 2 +/- 2 ng/ml), and caused degranulation of cytochalasin B pretreated human neutrophils (ED50 of 20 ng/ml). In purified human monocytes, chemotactic responses to LYNAP at doses up to 100 ng/ml were absent, indicating nonidentity with a lymphocyte-derived monocyte chemotactic factor previously described by other workers. LYNAP shows biochemical and biologic similarities to a recently detected monocyte-derived neutrophil-activating peptide (MONAP). Moreover, desensitization experiments revealed cross-deactivation between LYNAP and MONAP, not, however, between these two chemotactic peptides and other well characterized polymorphonuclear leukocyte chemotaxins, e.g., C5a, FMLP, leukotriene B4, or platelet-activating factor. This finding points toward structure identity or homology of both chemotaxins, MONAP and LYNAP.     

IL-9 inhibits oxidative burst and TNF-alpha release in lipopolysaccharide-stimulated human monocytes through TGF-beta

IL-9 is a Th2 cytokine that exerts pleiotropic activities on T cells, B cells, mast cells, hematopoietic progenitors, and lung epithelial cells, but no effect of this cytokine has been reported so far on mononuclear phagocytes. Human blood monocytes preincubated with IL-9 for 24 h before LPS or PMA stimulation exhibited a decreased oxidative burst, even in the presence of IFN-gamma. The inhibitory effect of IL-9 was specifically abolished by anti-hIL-9R mAb, and the presence of IL-9 receptors was demonstrated on human blood monocytes by FACS. IL-9 also down-regulated TNF-alpha and IL-10 release by LPS-stimulated monocytes. In addition, IL-9 strongly up-regulated the production of TGF-beta1 by LPS-stimulated monocytes. The suppressive effect of IL-9 on the respiratory burst and TNF-alpha production in LPS-stimulated monocytes was significantly inhibited by anti-TGF-beta1, but not by anti-IL-10Rbeta mAb. Furthermore, IL-9 inhibited LPS-induced activation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases in monocytes through a TGF-beta-mediated induction of protein phosphatase activity. In contrast, IL-4, which exerts a similar inhibitory effect on the oxidative burst and TNF-alpha release by monocytes, acts primarily through a down-regulation of LPS receptors. Thus, IL-9 deactivates LPS-stimulated blood mononuclear phagocytes, and the mechanism of inhibition involves the potentiation of TGF-beta1 production and extracellular signal-regulated kinase inhibition. These findings highlight a new target cell for IL-9 and may account for the beneficial activity of IL-9 in animal models of exaggerated inflammatory response.     

Mitogenic activation of human T cells induces two closely related genes which share structural similarities with a new family of secreted factors

Previously we have isolated about 60 novel cDNA clones whose corresponding mRNAs are induced by mitogenic activation in human peripheral blood T cells. Here we describe the primary structure and regulation of two such cloned genes, pAT 464 and pAT 744, which may encode new lymphokines/cytokines. Similar to IL-2, both genes require the synergy of agents such as PHA and PMA for optimal expression, and, in addition, the induction of both is sensitive to the immunosuppressive drug cyclosporin A. The two genes can be expressed in T cells, B cells, and the promyelocytic cell line HL60, but they are not expressed in human fibroblasts, suggesting that their expression is restricted to hematopoietic lineages. The predicted peptides encoded by these two clones feature hydrophobic N-terminal leaders characteristic of secreted proteins. The predicted size of both proteins is about 8 kDa upon cleavage of the putative leader peptide. pAT 464 and pAT 744 are very similar to each other and also share some critical amino acid similarity with a newly emerging family of secreted factors including connective tissue activating factor III, platelet factor 4, an IFN-gamma-induced factor, macrophage inflammatory protein, and a factor chemotactic to neutrophils (3-10C, monocyte-derived neutrophil chemotactic factor, neutrophil-activating factor). Some of these factors have been shown to display functions associated with an inflammatory response and/or have mitogenic activities. Collectively, the data presented here suggest that pAT 464 and pAT 744 encode novel lymphokines/cytokines which may play roles during an immune response similar to those enacted by these structurally related factors.     

Modulation of natural and interleukin-2-induced tumour-cytolytic activities by the members of a protein family related to β-thromboglobulin

Summary A preparation of three C-terminal fragments of the platelet protein -thromboglobulin was previously described to have immunomodulatory properties on phagocytic cells. One of the components is obviously identical to the recently described neutrophil-activating peptide 2 (NAP-2). In further investigations on this protein preparation (called factor C) we are able to show an additional influence on the tumour-cytolytic activities of mononuclear cells. Total neutralization of the factor C effect, by treating a factor C preparation with specific monoclonal antibody C24 prior to application in cell culture, proved that the effect is really restricted to factor C proteins. If factor C is given in combination with natural interleukin-2 (IL-2) a dose-dependent suppression of IL-2-mediated natural killer lymphokine-activated killer activity can be measured, which is first detectable 72 h after addition of factor C. Suppression does not occur if the both factors are added within a time interval of more than 12 h. Depletion of monocytes from mononuclear cells has no effect on factor-C-mediated cytotoxicity, demonstrating that factor C acts directly on lymphoid cells.     

Cilomilast counteracts the effects of cigarette smoke in airway epithelial cells

Cigarette smoke extracts (CSE) alter TLR4 expression and activation in bronchial epithelial cells. Cilomilast, a phosphodiesterase-4 inhibitor, inhibits cigarette smoke-induced neutrophilia.This study was aimed to explore whether cilomilast, in a human bronchial epithelial cell line (16-HBE), counteracted CSE effects. In particular, TLR4 expression, IP-10 and IL-8 release, lymphocyte and neutrophil chemotactic activity and ERK and IkBa phosphorylation in CSE and LPS-stimulated 16-HBE were assessed.CSE increased TLR4 expression, reduced IP-10 release and lymphocyte chemotactic activity and increased IL-8 release and neutrophil chemotactic activity. Cilomilast reduced TLR4 expression, IL-8 release and neutrophil chemotactic activity as well as it increased IP-10 release and lymphocyte chemotactic activity. All these cilomilast mediated effects were associated with a reduced ERK1/2 and with an increased IkBa phosphorylation.In conclusion, the present study provides compelling evidences that cilomilast may be considered a possible valid therapeutic option in controlling inflammatory processes present in smokers.     

Human neutrophil Fc gamma RIIIB and formyl peptide receptors are functionally linked during formyl-methionyl-leucyl-phenylalanine-induced chemotaxis.

The formyl peptide receptor (FPR) and the glycosyl-phosphatidylinositol-linked type III receptor for the Fc portion of IgG (Fc gamma RIIIB; CD16) play important roles in various inflammatory responses in human neutrophils. The mechanisms of signaling by the glycosyl phosphatidylinositol-anchored Fc gamma RIIIB are not known. Therefore, we investigated the possibility that Fc gamma RIIIB and FPR may act in concert to mediate neutrophil functions. We observed that pretreatment of normal human neutrophils with Fab fragments of a mAb to the Fc gamma RIII (3G8) specifically inhibited their chemotaxis into micropore filters in response to the formylated peptides FMLP or formyl-norleucyl-leucyl-phenylalanine. Pretreatment of neutrophils with a saturating concentration of 3G8 Fab (100 nM or 5 micrograms/ml) followed by exposure to FMLP (0.5 to 500 nM) indicated that significant inhibition of chemotaxis was observed at peptide concentrations greater than 5 nM. However, 3G8 Fab had no effect on the neutrophil response to a wide range (0.05 to 500 nM) of other chemotactic factors, including C5a, leukotriene B4, IL-8 (neutrophil-activating peptide-1), and platelet-activating factor. Moreover, pretreatment of neutrophils with mAb to other cell surface molecules (decay-accelerating factor, Fc gamma RII, and HLA class I) did not affect chemotaxis to FMLP. Inhibition of movement was not due to degradation of FMLP by the cell surface endopeptidase 24.11 (CD10), because neutrophils pretreated with the CD10 inhibitor phosphoramidone and 3G8 Fab displayed the same altered response to FMLP as cells pretreated with 3G8 Fab alone. Ligation of the Fc binding site of Fc gamma RIIIB appears to be essential for altering the FMLP-induced response, since soluble aggregated IgG and other anti-Fc gamma RIII antibodies, all of which recognize the ligand binding site, mimic the inhibitory effect of the 3G8 Fab on FMLP-induced chemotaxis. In contrast, a mAb (214.1) that does not recognize the Fc binding site of Fc gamma RIIIB had no effect on FMLP-induced chemotaxis. Not only did anti-Fc gamma RIII inhibit neutrophil chemotaxis to FMLP in a filter-based migration assay, but 3G8 Fab also inhibited FMLP-induced neutrophil transendothelial migration. Scatchard plot analysis of radioligand binding experiments indicated that 3G8 Fab did not significantly alter the number of FMLP binding sites on neutrophils but significantly increased the affinity of the FPR for [3H]FMLP. Removal of greater than 80% of cell surface Fc gamma RIIIB by phospholipase C abolished the neutrophil chemotactic response to FMLP but did not affect movement toward C5a, IL-8, or leukotriene B4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential effects of neutrophil-activating peptide 1/IL-8 and its homologues on leukocyte adhesion and phagocytosis.

Several structural homologues of the chemotactic peptide neutrophil-activating peptide 1/IL-8 (NAP-1/IL-8) were tested for their ability to influence the expression and function of adhesion-promoting receptors on human polymorphonuclear leukocytes (PMN). NAP-2, melanoma growth stimulatory activity, and two forms of NAP-1/IL-8 (ser-NAP-1/IL-8 and ala-NAP-1/IL-8, consisting of 72 and 77 amino acids, respectively), each caused an increase in the expression of CD11b/CD18 (CR3) and CR1, which was accompanied by a decrease in the expression of leukocyte adhesion molecule-1 (LAM-1, LECAM-1). The binding activity of CD11b/CD18 was also enhanced 3- to 10-fold by these peptides, but enhanced function was transient: binding of erythrocytes coated with C3bi reached a maximum by 30 min and declined thereafter. Ser-NAP-1/IL-8, ala-NAP-1/IL-8, NAP-2, and melanoma growth stimulatory activity also caused a two- to threefold enhancement of the phagocytosis of IgG-coated erythrocytes (EIgG) by PMN without causing a large increase in the expression of Fc gamma receptors. Enhanced phagocytosis of EIgG appeared to be mediated through CD11b/CD18, because F(ab')2 fragments of an antibody directed against CD18 inhibited NAP-1/IL-8-stimulated ingestion of EIgG. The four active peptides caused a rapid, transient increase in the amount of F-actin within PMN, indicating that they are capable of influencing the structure of the microfilamentous cytoskeleton, which participates in phagocytosis. Two other NAP-1/IL-8-related peptides, platelet factor 4 and connective tissue-activating peptide III, were without effect on expression of CD11b/CD18, CR1, and LAM-1, binding activity of CD11b/CD18, or Fc-mediated phagocytosis, and increased actin polymerization only slightly. Our observations indicate that several members of the NAP-1/IL-8 family of peptides were capable of promoting integrin-mediated adhesion and Fc-mediated phagocytosis, processes important in the recruitment of PMN to sites of inflammation and antimicrobial responses of PMN.     

Expression of IgG Fc receptors in myeloid leukemic cell lines. Effect of colony-stimulating factors and cytokines

Three classes of FcR have been defined on human myeloid cells by their reactivity with mAb; FcRI (mAb 32); FcRII (mAb IV3); and FcRIII (mAb 3G8). We have quantitated the expression of each FcR on human myeloid leukemia cells and cell lines (KG-1, HL-60, U937, and K562). Detailed analysis of FcR surface expression is provided for the U937 cell line after exposure to CSF and cytokines. Increased expression of FcRI and FcRII occurred at 72 h in cells exposed to GCT or Mo cell line-conditioned medium as well as to medium from PHA-treated mononuclear cells. The augmentation of FcRII required protein synthesis and was diminished by a neutralizing antibody to granulocyte-macrophage CSF. We also show that fractions containing natural granulocyte CSF or granulocyte-macrophage CSF as well as r-granulocyte and r-granulocyte-macrophage CSF are capable of inducing FcRII on these cells, whereas other cytokines such as IL-1 and IL-2, TNF-alpha, INF-gamma and macrophages CSF failed to do so.     

Neutrophil chemotactic factor produced by lipopolysaccharide (LPS)-stimulated human blood mononuclear leukocytes: partial characterization and separation from interleukin 1 (IL 1)

LPS stimulated human blood mononuclear leukocytes to produce a chemotactic factor for human neutrophils. The effect of LPS was dose-dependent; 10 micrograms/ml was optimal for production of chemotactic factor. Chemotactic activity was detected 3 hr after LPS stimulation, and reached its peak at 12 hr. No activity was detected in culture supernatants of unstimulated cells, provided LPS-free media were selected. Isoelectric point of the factor, determined by chromatofocusing, was approximately 8 to 8.5. Molecular weight was approximately 10 kilodaltons by Sephacryl S-200 gel filtration or by HPLC gel filtration on TSK-2000 and -3000 columns in succession. The gel filtration fractions were also assayed for IL 1 activity. The elution position of IL 1 activity corresponded to a m.w. of 18. There was no chemotactic activity in the IL 1 activity peak. Furthermore, highly purified natural Il 1 alpha and -beta and recombinant Il 1 alpha and -beta did not exhibit chemotactic activity for neutrophils in our assay. Among mononuclear leukocytes, the monocyte was the principal producer of neutrophil chemotactic factor. These results suggest that a chemotactic factor for neutrophils, different from IL 1, is produced by LPS-stimulated blood monocytes.     

Differential regulation of neutrophil-activating chemokines by IL-6 and its soluble receptor isoforms

Interleukin-6 signaling via its soluble receptor (sIL-6R) differentially regulates inflammatory chemokine expression and leukocyte apoptosis to coordinate transition from neutrophil to mononuclear cell infiltration. sIL-6R activities may, however, be influenced in vivo by the occurrence of two sIL-6R isoforms that are released as a consequence of differential mRNA splicing (DS) or proteolytic cleavage (PC) of the cognate IL-6R (termed DS- and PC-sIL-6R). Using human peritoneal mesothelial cells and a murine model of peritoneal inflammation, studies described in this work have compared the ability of both isoforms to regulate neutrophil recruitment. In this respect, DS- and PC-sIL-6R were comparable in their activities; however, these studies emphasized that IL-6 trans signaling differentially controls neutrophil-activating CXC chemokine expression. In vitro, stimulation of mesothelial cells with IL-6 in combination with either DS-sIL-6R or PC-sIL-6R showed no induction of CXC chemokine ligand (CXCL)1 (GRO alpha) and CXCL8 (IL-8), whereas both isoforms enhanced CXCL5 (ENA-78) and CXCL6 (granulocyte chemotactic protein-2) expression. Moreover, when complexed with IL-6, both isoforms specifically inhibited the IL-1 beta-induced secretion of CXCL8. These findings were paralleled in vivo, in which induction of peritoneal inflammation in IL-6-deficient (IL-6(-/-)) mice resulted in enhanced keratinocyte-derived chemokine and macrophage-inflammatory protein-2 (the murine equivalent of CXCL1 and CXCL8) levels, but reduced LPS-induced CXC chemokine (the murine equivalent of CXCL5) expression. Reconstitution of IL-6 signaling in IL-6(-/-) mice with IL-6 and its soluble receptor isoforms corrected this chemokine imbalance and suppressed overall neutrophil infiltration. These data confirm that sIL-6R-mediated signaling primarily limits neutrophil influx; however, induction of CXCL5 and CXCL6 may regulate other neutrophil responses.