Beta 2 integrin-dependent tyrosine phosphorylation of paxillin in human neutrophils treated with tumor necrosis factor

The focal adhesion protein paxillin undergoes tyrosine phosphorylation in response to signals mediated by integrins, neuropeptides and oncogene products, possibly via activation of the focal adhesion- associated kinase, p125FAK. In the present work, tumor necrosis factor- alpha (TNF) stimulated tyrosine phosphorylation of paxillin in human neutrophils. Cell adhesion and participation of the beta 2 integrin CD18 were necessary, but not sufficient, for the response. Adherent neutrophils also tyrosine phosphorylated paxillin in response to phorbol ester, formylmethionyl-leucyl-phenylalanine and opsonized bacteria. In contrast, p125FAK was constitutively tyrosine phosphorylated in a manner unaffected by adherence and/or TNF. Thus, cytokines and microbial products are among the stimuli that can induce the tyrosine phosphorylation of paxillin, and kinases other than p125FAK may be responsible. This is the first identification of paxillin and p125FAK in human cells and neutrophils, and one of the few identifications of a specific protein that undergoes tyrosine phosphorylation in response to any agonist in neutrophils or in response to TNF in any cell.     

Beta 2 integrin-dependent protein tyrosine phosphorylation and activation of the FGR protein tyrosine kinase in human neutrophils

Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigated the dependence of this response on beta 2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express beta 2 integrins, and by plating PMN on surface bound anti-beta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF. Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti- Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58fgr) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58fgr in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of beta 2 integrins such as phorbol-12- myristate 13-acetate (PMA), and formyl methionyl-leucyl-phenylalanine (FMLP), induced activation of p58fgr kinase activity. Activation of p58fgr kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN, beta 2 integrins are implicated in triggering of protein tyrosine phosphorylation, and establish a link between beta 2 integrin-dependent adhesion and the protein tyrosine kinase fgr in cell signaling.     

Adhesion-dependent protein tyrosine phosphorylation in neutrophils treated with tumor necrosis factor

Human neutrophils (PMN) respond to tumor necrosis factor (TNF) by releasing their granules, reorganizing their cytoskeleton, and massively secreting hydrogen peroxide. This response is dependent on adhesion to extracellular matrix proteins and expression of CD11b/CD18 integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. D. Wright. 1989. J. Cell Biol. 109:1341-1349). We investigated the role of tyrosine phosphorylation in the response of PMN to TNF. PMN adherent to protein-coated surfaces but not suspended PMN showed tyrosine phosphorylation of several proteins (approximately 150, approximately 115, approximately 75, and approximately 65 kD) in response to TNF. Tyrosine phosphorylation was evident 5 min after addition of TNF and lasted at least 2 h. The tyrosine kinase inhibitors K252a, genistein and ST638 suppressed tyrosine phosphorylation and blocked hydrogen peroxide production in a reversible manner at low concentrations. Tyrosine kinase inhibitors also blocked the spreading of PMN in response to TNF. Dihydrocytochalasin B did not inhibit tyrosine phosphorylation, but in its presence phosphorylation was rapidly reversed. By immunocytochemistry, the majority of tyrosine phosphoproteins were localized to focal adhesions. Thus TNF-induced tyrosine phosphorylation depends on adhesion of PMN to extracellular matrix proteins, and participates in the transduction of the signals that direct the cells to spread on a biological surface and undergo a respiratory burst.     

Serine phosphorylation of p60 tumor necrosis factor receptor by PKC-delta in TNF-alpha-activated neutrophils

Tumor necrosis factor-(TNF-) triggers degranulation and oxygen radical release in adherentneutrophils. The p60TNF receptor (p60TNFR) is responsible forproinflammatory signaling, and protein kinase C (PKC) is a candidatefor the regulation of p60TNFR. Both TNF- and the PKC-activatorphorbol 12-myristate 13-acetate triggered phosphorylation of p60TNFR.Receptor phosphorylation was on both serine and threonine but not ontyrosine residues. The PKC- isotype is a candidate enzyme for serinephosphorylation of p60TNFR. Staurosporine and the PKC- inhibitorrottlerin inhibited TNF--triggered serine but not threoninephosphorylation. Serine phosphorylation was associated withreceptor desensitization, as inhibition of PKC resulted in enhanceddegranulation (elastase release). After neutrophil activation, PKC-was the only PKC isotype that associated with p60TNFR within thecorrect time frame for receptor phosphorylation. In vitro, onlyPKC-, but not the -, I-, II-, or -isotypes, wascompetent to phosphorylate the receptor, indicating that p60TNFR is adirect substrate for PKC-. These findings suggest a selective rolefor PKC- in negative regulation of the p60TNFR and ofTNF--induced signaling.

     

Stimulation of neutrophils by tumor necrosis factor

Human recombinant tumor necrosis factor (TNF) was shown to be a weak direct stimulus of the neutrophil respiratory burst and degranulation. The stimulation, as measured by iodination, H2O2 production, and lysozyme release, was considerably increased by the presence of unopsonized zymosan in the reaction mixture, an effect which was associated with the increased ingestion of the zymosan. TNF does not act as an opsonin but, rather, reacts with the neutrophil to increase its phagocytic activity. TNF-dependent phagocytosis, as measured indirectly by iodination, is inhibited by monoclonal antibodies (Mab) 60.1 and 60.3, which recognize different epitopes on the C3bi receptor/adherence-promoting surface glycoprotein of neutrophils. Other neutrophil stimulants, namely N-formyl-methionyl-leucyl-phenylalanine, the Ca2+ ionophore A23187, and phorbol myristic acetate, also increase iodination in the presence of zymosan; as with TNF, the effect of these stimulants is inhibited by Mab 60.1 and 60.3, whereas, in contrast to that of TNF, their stimulation of iodination is unaffected by an Mab directed against TNF. TNF may be a natural stimulant of neutrophils which promotes adherence to endothelial cells and to particles, leading to increased phagocytosis, respiratory burst activity, and degranulation.     

Recombinant human tumor necrosis factor alpha lacks chemotactic activity for human peripheral blood neutrophils and monocytes

Preparations of recombinant human tumor necrosis factor alpha (rhuTNF alpha) free of aminoterminal methionine were tested for human neutrophil granulocyte (PMN) and monocyte (MO) chemotactic activity using the Boyden chamber system. Over a wide range of concentrations (10(-7)-10(-15) M) rhuTNF alpha of two different sources failed to elicit chemotactic responses in PMN or MO, whereas strong PMN and MO chemotactic activity could be detected using the tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). In addition, rhuTNF alpha containing 62% aminoterminal methionine failed to induce PMN and MO chemotaxis. It is concluded that rhuTNF alpha may not be a chemotaxin for human PMN and MO in vitro.     

Inactivation of recombinant human tumor necrosis factor-alpha by proteolytic enzymes released from stimulated human neutrophils

Activated human neutrophils (PMN) degrade rTNF-alpha resulting in a loss of cytotoxic activity against murine L-929 cells (L cells). This inactivation is mediated through proteases released from activated PMN. Exposure of TNF to H2O2, glucose oxidase, xanthine oxidase, or myeloper-oxidase-H2O2-halide did not affect TNF cytotoxicity for L cells. Exposure to trypsin, chymotrypsin, pronase E, or elastase, however, did diminish TNF bioactivity. FMLP-stimulated PMN in the presence, but not in the absence, of cytochalasin B reduced TNF activity, whereas PMA-stimulated PMN did not affect TNF. Stimulation of PMN with opsonized bacteria also induced TNF inactivation as well as the supernatant of FMLP-stimulated cells. Addition of protease inhibitors to the FMLP-stimulated cytochalasin B-treated PMN abrogated the inactivation of TNF cytotoxicity for L cells, whereas scavengers were not protective. In addition, PMN from a chronic granulomatous disease patient also decreased TNF bioactivity. Inactivation of TNF by activated PMN correlated with granule release and not with superoxide production. Exposure of TNF to proteases and FMLP-activated PMN also resulted in a loss of reactivity with anti-TNF antibodies, as measured by ELISA, and in the formation of an approximately 10-kDa split product from the 17-kDa rTNF molecule. Partial degradation of TNF by proteases released from activated PMN may result in a diminished TNF bioactivity and thereby contribute to the regulation of local inflammatory reactions.     

Binding of human tumor necrosis factor alpha to multimeric complementary peptides.

A peptide with binding properties for tumor necrosis factor (TNF alpha) sequence 144-157 has been designed, using a computer-assisted method able to create peptide sequences hydropathically complementary to a given sequence. The complementary peptide was synthesized in a multimeric form starting from an octadentate polylysine core, to facilitate its immobilization and to provide interaction multivalency. Once immobilized on a solid support to prepare an affinity column, it recognized the target TNF144-157 peptide selectively from crude peptide mixtures containing TNF fragments encompassing the entire TNF alpha sequence. Similar selectivity and specificity were shown for full-length recombinant TNF alpha, allowing its purification from crude Escherichia coli extracts. The octameric complementary peptide preserved its recognition properties for TNF alpha and biotinylated TNF alpha even after coating on microtiter plates. Competitive binding occurred with unlabeled TNF alpha in the range between 0.01 and 10 micrograms/ml, in the presence of detergent such as 0.05% Tween 20 and in the presence of 1% normal goat serum. The effect of complementary peptide multimerization was evidenced by its enhanced binding affinity for TNF alpha, which exists in solution as a trimer, while the target TNF[144-157] peptide was recognized with much lower strength. The dissociation constant for interaction with TNF alpha was close to 10 nM, allowing its easy detection by solid phase assays in concentrations as low as 10 pmol/ml.     

Tumor necrosis factor modulates epidermal growth factor receptor phosphorylation and kinase activity in human tumor cells. Correlation with cytotoxicity

Tumor necrosis factor (TNF) is a cytokine which induces cytotoxicity in some but not all tumor cells. Initial studies of five tumor cell lines demonstrated that TNF was able to rapidly (within 30 min) modulate tyrosine protein kinase activity of epidermal growth factor (EGF) receptors on tumor cell lines which were sensitive to the cytotoxic effects of TNF but not alter EGF receptor kinase activity in TNF-resistant tumor cells. Two tumor cell lines (ME-180 cervical carcinoma and T24 bladder carcinoma) which have been shown to express similar TNF-binding characteristics but differ in their sensitivity to the cytotoxic actions of TNF were chosen for further characterization. Treatment of TNF-sensitive ME-180 cells with 1 nM TNF resulted in a 3-fold stimulation of EGF receptor tyrosine protein kinase activity within 10 min which correlated with increased phosphorylation of EGF receptor protein itself. In addition, dose-response studies indicate that similar concentrations of TNF modulate both ME-180 cell growth and EGF receptor kinase activity. Treatment of TNF-resistant T24 cells showed that TNF had no significant effect on their growth, EGF receptor tyrosine protein kinase activity, or phosphorylation of EGF receptor protein although EGF receptor kinase activity was stimulated by EGF. Quantitation of receptors expressed on the surface of ME-180 and T24 cells demonstrated a 3-fold difference between the number of EGF-binding sites on T24 (100,000) versus ME-180 cells (300,000), suggesting the relative abundance of EGF receptor does not solely account for differential effects of TNF on EGF receptor activation in these two cell lines. Phosphoamino acid analysis of EGF receptor from 32P-equilibrated ME-180 cells demonstrated that TNF-induced phosphorylation of amino acids which was quantitatively similar to that of EGF but distinct from the effects of phorbol ester. However, unlike EGF, TNF was unable to stimulate EGF receptor kinase activity in ME-180 cell lysates. The kinetics of EGF receptor activation and the metabolic consequence of activation of EGF receptor activity by TNF appear to be distinct from those induced by EGF. These results suggest that TNF-induced modulation of EGF receptor occurs through a unique mechanism and may play a role in the cytotoxic actions of TNF.     

Novel muteins of human tumor necrosis factor alpha

For chemical synthesis of a gene coding for human tumor necrosis factor alpha (TNF-alpha), DNA sequence predicted by the amino acid sequence of human TNF molecule was prepared. Codons were chosen according to the codon usage in Escherichia coli (E. coli). The 490 bp gene was assembled by enzymic ligation of 42 oligonucleotides and was cloned into a vector (pKK223-3) for high expression of active TNF-alpha in E. coli. With use of site-directed mutagenesis on this DNA, five different muteins of TNF-alpha were synthesized. TNF-M1 and TNF-M4 have deletions of His-73 and Gln-102, respectively. These deletions didn't cause loss of the cytotoxic activity against L929 cells. TNF-M5, which has a substitution of Asp-10 to Arg, had the similar cytotoxic activity to that of TNF-alpha. The cytotoxic spectra against several tumor cells were not changed by this substitution. TNF-M3 has an amino acid substitution of Glu-116 to His which occupies this position in human TNF-beta. This substitution didn't change the cytotoxicity. In addition, evidence was presented that the change of the carboxyl terminal residue doesn't always influence the cytotoxic activity of TNF-alpha. Many different muteins were also isolated by random mutagenesis with hydroxylamine-HCl. One of the muteins, which carries a mutation of His-15 to Tyr, lost the cytotoxic activity almost completely.     

Bradykinin upregulates IL-8 production in human gingival fibroblasts stimulated by interleukin-1beta and tumor necrosis factor alpha

The proinflammatory mediator bradykinin (BK) is suggested to play an important role in the pathogenesis of various inflammatory diseases including periodontitis. In this study, BK per se stimulated interleukin-8 (IL-8) production in human gingival fibroblasts in vitro. Furthermore, BK upregulated the stimulatory effect of the cytokines IL-1beta and TNFalpha on the production of IL-8. The stimulatory effect of BK on the IL-1beta- or TNFalpha-stimulated IL-8 production was reduced in the presence of BK B2 receptor antagonist HOE 140, whereas the B1 receptor antagonist Lys-(des-arg9, Leu8)-BK had no effect. Similar to BK, the calcium ionophore A23187 also upregulated the stimulatory effect of IL-1beta and TNFalpha on IL-8 production. The protein kinase C (PKC) inhibitor bisindolylmaleimide, BIS, significantly reduced the stimulatory effect of BK on IL-1beta and TNFalpha increased IL-8 production but did not affect the production of IL-8 stimulated by cytokines alone. The specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 reduced IL-8 production stimulated by the combination of BK and IL-1beta as well as the IL-1beta-stimulated IL-8 production. In conclusion, this study shows that BK upregulates IL-1beta- and TNFalpha-stimulated IL-8 production via BK B2 receptor and that PKC signal pathway seems to be involved in the upregulation of the cytokine-induced IL-8 production in gingival fibroblasts. This stimulatory effect of BK on IL-8 production may contribute to the maintenance of the gingival inflammation and enhanced risk for destruction of gingival connective tissue.     

Correlation between spontaneous oscillations of cytosolic free Ca2+ and tumor necrosis factor-induced degranulation in adherent human neutrophils

By using a hemolytic plaque assay to detect release of lactoferrin and myeloperoxidase, tumor necrosis factor (TNF) was shown previously to induce secretion of these granule proteins from single adherent neutrophils. Secretion was inhibited by loading neutrophils with calcium chelators, indicating a crucial role of cytosolic free [Ca2+] in the signal transduction mechanism of TNF. In the present study, using a microfluorometer technique to follow changes in the cytosolic free [Ca2+] in single adherent neutrophils, we were not able to detect any TNF-induced [Ca2+] transients. However, these adherent cells exhibited spontaneous oscillations of their cytosolic free [Ca2+], as previously reported (Jaconi, M.E.E., Rivest, R.W., Schlegel, W., Wollheim, C.B., Pittet, D., and Lew, P.D. (1988) J. Biol. Chem. 263, 10557-10560). A close correlation was found between a reduced oscillatory activity of cytosolic free [Ca2+] and a reduced ability of TNF to induce degranulation, by reducing the extracellular [Ca2+] or loading the cells with a calcium chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). In addition, when the cells were incubated at 37 degrees C for 3 h there was a parallel decline in the spontaneous oscillatory activity of cytosolic free [Ca2+] and TNF-induced secretion of lactoferrin. Control experiments showed that phorbol 12-myristate 13-acetate-induced secretion was not affected under the same conditions, indicating that the secretory process per se was not disturbed. We conclude that TNF by itself does not give rise to any changes of the cytosolic free [Ca2+] but that the spontaneous oscillatory activity of cytosolic free [Ca2+] in adherent neutrophils is necessary for TNF-induced degranulation.     

Determination of human tumor necrosis factor alpha by a highly sensitive enzyme immunoassay.

Tumor necrosis factor alpha (TNF-alpha) is a polypeptide produced primarily by monocytes and macrophages. It is involved in a wide variety of immune reactions. A simple and sensitive microplate enzyme-linked immunosorbent assay for the detection of hTNF-alpha in serum, plasma, and cell culture supernatants is described. The method is based on the use of horseradish peroxidase in biotin-streptavidin amplification system which is performed in Nunc StarWell. This system has enabled us to achieve a sensitivity of 0.1 pg hTNF-alpha/ml of the sample. The assay is calibrated to the World Health Organization (WHO) standard for hTNF-alpha (87/650). The within-run coefficient of variation ranged from 3.7 to 5.9 and the between-run coefficient of variation ranged from 8.0 to 9.9. The results obtained by the proposed method and by a commercially available kit (DRG hTNF-alpha ELISA) correlated well (n = 20, r = 0.956).     

Monoclonal antibodies to human tumor necrosis factor alpha: in vitro and in vivo application.

Three stable murine hybridoma cell lines, which secrete monoclonal antibodies (mAb) to human tumor necrosis factor alpha (TNF alpha), were established. None of the monoclonal antibodies cross-reacted with lymphotoxin, interleukin 2 (IL 2) or Interferon gamma (IFN gamma). The highly species-specific monoclonal antibody, designated as mAb 195, neutralizes the cytotoxic activity of human and chimpanzee TNF alpha. This antibody was further used during in vivo studies to neutralize human TNF alpha in a murine animal model. The mAb 114 is a weakly neutralizing antibody that binds to a different epitope of TNF alpha than mAb 195. mAb 114 shows a wide range of cross-reactivity with TNF alpha of the following species: dog, pig, cynomolgus, rhesus, baboon and chimpanzee. The mAb 199 binds to human TNF alpha, but does not neutralize the cytotoxic activity. The epitope recognized by this mAb is in close proximity to mAb 114. A reproducible, sensitive immunoassay for human TNF7 alpha has been developed using the antibodies mAb 199 and mAb 195. The test is performed within 6 hr and detects TNF7 alpha in serum samples, with a limit of detection of 5 to 10 pg/mL.     

Activation of Jun N-terminal kinase/stress-activated protein kinase pathway by tumor necrosis factor alpha leads to intercellular adhesion molecule-1 expression.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine implicated in the pathogenesis of numerous chronic and acute inflammatory conditions. We have previously shown that mouse Sertoli cells respond to TNF-alpha by increasing interleukin-6 production and intercellular adhesion molecule-1 (ICAM-1) expression (1). In this cell type TNF-alpha activates the mitogen-activated protein kinase (MAPK) pathways p42/p44 MAPK, JNK/SAPK, and p38, the last of which is responsible for interleukin-6 production (1). To determine which MAPK signaling pathway is required for TNF-alpha induction of ICAM-1 expression, we have utilized the protein kinase inhibitor dimethylaminopurine, demonstrating that treatment of Sertoli cells with such compound significantly reduced ICAM-1 expression and JNK/SAPK activation. Moreover, dimethylaminopurine treatment increased the expression of MAPK phosphatase-2, providing a possible mechanism of action of this compound. By using agonist antibodies to p55 and to p75 TNF-alpha receptors and both human and mouse TNF-alpha, we demonstrate that both TNF receptors are expressed and that only the p55 receptor is involved in ICAM-1 expression. The p55 receptor activates all of the three pathways, whereas p75 failed to activate any of the MAPKs. Altogether our results demonstrate that TNF-alpha up-regulates ICAM-1 expression through the activation of the JNK/SAPK transduction pathway mediated by the p55 receptor.     

Growth inhibition of Candida albicans by human polymorphonuclear neutrophils: activation by interferon-gamma and tumor necrosis factor

This study was designed to determine whether anti-fungal activity in human polymorphonuclear neutrophils (PMN) might be under the regulation of cytokines such as tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma). By using a radiolabel microassay developed in our laboratory that makes use of the incorporation of [3H]glucose into residual candida, we demonstrated that PMN were better able to inhibit Candida albicans growth in vitro than peripheral blood lymphocytes (PBL). PMN from normal volunteers added to C. albicans for 24 hr at 37 degrees C in a 96-well microplate inhibited fungal growth almost completely at the 300:1 effector/target ratio and frequently at 100:1. Significant activity was still detected at 10:1. In contrast, PBL from the same donors had less activity than PMN at all the ratios tested and lost all function at the 30:1 ratio. TNF and IFN-gamma added to the PMN/candida cultures additionally enhanced PMN to inhibit candida growth. Both cytokines effectively activated PMN down to 0.1 to 0.01 U/ml, and neither cytokine interfered directly with fungal growth, even up to 1000 U/ml. Concentrations of TNF and IFN-gamma below the level that enhanced PMN function when added together to PMN acted synergistically to significantly enhance their anti-fungal activity. Therefore, TNF and IFN-gamma which are active on lymphoid cells, also appear to have the ability to directly activate PMN, and the synergistic action of the two cytokines at low doses that may be below the toxic range may prove to be of clinical importance in protection of immunocompromised host against opportunistic infections.