Search PubMed⌕ Search

Biomedical subjects

M A Vadas

Publications and source records attributed to M A Vadas.

At least 73 records · Page 4Linked to original sources
PubMed1994

A sequence-specific single-strand DNA binding protein that contacts repressor sequences in the human GM-CSF promoter.

NF-GMb is a nuclear factor that binds to the proximal promoter of the human granulocyte-macrophage colony stimulating factor (GM-CSF) gene. NF-GMb has a subunit molecular weight of 22 kDa, is constitutively expressed in embryonic fibroblasts and binds to sequences within the adjacent CK-1 and CK-2 elements (CK-1/CK-2 region), located at approximately -100 in the GM-CSF gene promoter. These elements are conserved in haemopoietic growth factor (HGF) genes. NF-GMb binding requires the presence of repeated 5'CAGG3' sequences that overlap the binding sites for positive activators. Surprisingly, NF-GMb was found to bind solely to single-strand DNA, namely the non-coding strand of the GM-CSF CK-1/CK-2 region. NF-GMb may belong to a family of single-strand DNA binding (ssdb) proteins that have 5'CAGG3' sequences within their binding sites. Functional analysis of the proximal GM-CSF promoter revealed that sequences in the -114 to -79 region of the promoter containing the NF-GMb binding sites had no intrinsic activity in fibroblasts but could, however, repress tumour necrosis factor-alpha (TNF-alpha) inducible expression directed by downstream promoter sequences (-65 to -31). Subsequent mutation analysis showed that sequences involved in repression correlated with those required for NF-GMb binding.

Base Sequence↗
PubMed1994

P-selectin interacts with a beta 2-integrin to enhance phagocytosis.

P-selectin is an adhesion molecule for myeloid cells that seems to be essential for the development of cellular inflammatory responses. We show that adhesion of neutrophils to purified and recombinant P-selectin enhances the phagocytosis of unopsonized zymosan particles as judged by the number of cells ingesting particles (30.2 +/- 5.8 vs 14.5 +/- 4.0, p = 0.002) and the number of particles ingested per cell (percentage increase 58.3 +/- 4.4%. p = 0.0002). The enhanced phagocytosis was inhibited by Abs to CD18 or CD11b, suggesting that P-selectin alters beta 2-integrin function. The enhancement was only seen in the presence of cations allowing the integrin to assume a particular extracellular conformation. Furthermore, P-selectin, although not altering the total expression of CD18 on neutrophils, significantly increased the binding of mAb 24, which detects an activation-dependent epitope. Our results support a signaling role for P-selectin in influencing beta 2-integrin function.

Animals↗
PubMed1994

Specific human granulocyte-macrophage colony-stimulating factor antagonists.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hemopoietic growth factor and activator of mature myeloid cell function. We have previously shown that residue 21 in the first helix of GM-CSF plays a critical role in both biological activity and high-affinity receptor binding. We have now generated analogues of GM-CSF mutated at residue 21, expressed them in Escherichia coli, and examined them for binding, agonistic, and antagonistic activities. Binding experiments showed that GM E21A, E21Q, E21F, E21H, E21R, and E21K bound to the GM-CSF receptor alpha chain with a similar affinity to wild-type GM-CSF and had lost high-affinity binding to the GM-CSF receptor alpha-chain-common beta-chain complex. From these mutants, only the charge reversal mutants E21R and E21K were completely devoid of agonistic activity. Significantly we found that E21R and E21K antagonized the proliferative effect of GM-CSF on the erythroleukemic cell line TF-1 and primary acute myeloid leukemias, as well as GM-CSF-mediated stimulation of neutrophil superoxide production. This antagonism was specific for GM-CSF in that no antagonism of interleukin 3-mediated TF-1 cell proliferation or tumor necrosis factor alpha-mediated stimulation of neutrophil superoxide production was observed. E. coli-derived GM E21R and E21K were effective antagonists of both nonglycosylated and glycosylated wild-type GM-CSF. These results show that low-affinity GM-CSF binding can be dissociated from receptor activation and have potential clinical significance for the management of inflammatory diseases and certain leukemias where GM-CSF plays a pathogenic role.

Amino Acid Sequence↗
PubMed1994

Identification of residues in the first and fourth helices of human granulocyte-macrophage colony-stimulating factor involved in biologic activity and in binding to the alpha- and beta-chains of its receptor.

Residues within the first and fourth helices of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) were analyzed for their role in biologic activity and interaction with the alpha- and beta-chains of the hGM-CSF receptor. Within the first helix substitution of the surface residues Glu14, Asn17, Gln20, Arg23, Arg24, and Asn27 or the buried residues Ala18, Leu25, and Leu28 did not significantly impair bioactivity or receptor binding. Substitutions at the buried residues Ala22 and Leu26 had intermediate bioactivity. However, substitutions of the surface residue Glu21 or the buried residue Ile19 reduced the relative bioactivity of the analogues to as little as 0.45% and 0.3%, respectively. Substitution of the charged surface residues of the fourth helix showed that substitution at Glu104, Lys107, and Lys111 had no significant effect on bioactivity, but substitution at Glu108 and Asp112 reduced the potency of the analogues to 34% and 7%, respectively. Receptor binding studies showed that, whereas Glu21 is the critical residue for binding to the hGM-CSF-receptor beta-chain, Asp112 is likely to be involved in binding to the GM-CSF-receptor alpha-chain. These results establish the relative contribution of residues in the first and fourth helices for GM-CSF bioactivity and receptor binding, and support a model where the fourth helix of GM-CSF interacts with the alpha-chain, and the first helix with the beta-chain of the GM-CSF receptor.

Amino Acid Sequence↗
PubMed1994

Requirement for nuclear factor (NF)-kappa B p65 and NF-interleukin-6 binding elements in the tumor necrosis factor response region of the granulocyte colony-stimulating factor promoter.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor produced by mesenchymal and myeloid cells following activation by inflammatory stimuli. It has previously been shown that a region of the G-CSF promoter, (-200 to -165) containing the decanucleotide CK-1 element and two repeated sequences that resemble nuclear factor (NF)-interleukin-6 (IL-6) binding sites, is required for activation of the G-CSF gene by tumor necrosis factor-alpha (TNF-alpha) and IL-1 beta. We now show that the NF-kappa B p65 protein can bind to and activate this TNF response region. There are several unusual features of this p65 interaction with the TNF response region. First, NF-kappa B p65 but not the related NF-kappa B p50 binds to the CK-1 element and a p50/65 hybrid protein that relies on the p50 rel homology domain for DNA binding does not transactivate the TNF response region. Second, p65 transactivation of this region is cell specific and requires not only its own binding site but also the NF-IL6 consensus sites. NF-IL6 also binds to the TNF response region of the G-CSF promoter. Electrophoretic mobility shift studies show that p65 and NF-IL6 can bind cooperatively to the TNF response region. The ability of this region to respond to TNF-alpha or p65 is correlated with the ability to form the p65/NF-IL6 ternary complex.

Animals↗
PubMed1994

Two contiguous residues in human interleukin-3, Asp21 and Glu22, selectively interact with the alpha- and beta-chains of its receptor and participate in function.

We have previously reported that the predicted first helix of human interleukin (IL)-3 contains a hydrophilic region encompassing residues Asp21, Glu22, and Thr25 that is crucial for biological activity and IL-3 receptor binding. Using single amino acid substitution mutagenesis, we have now determined that Asp21 and Glu22, but not Thr25, were crucial for full IL-3 activity. Mutant D21R was 30-fold less potent than wild type IL-3 in the stimulation of biological activity. It also exhibited a similar reduction in its ability to bind to the cloned high affinity IL-3 receptor complex (alpha- and beta-chains) or to the receptor alpha-chain alone, indicating that residue 21 is involved in contacts with the alpha-chain. Mutant E22R was approximately 20,000-fold less potent than wild type IL-3 in the stimulation of biological activity and in binding to the IL-3 receptor high affinity complex. However, the binding of E22R to the IL-3 receptor alpha-chain alone was similar to that of wild type IL-3, suggesting that this mutant was defective in interactions with the receptor beta-chain. These results show that two contiguous residues in the N-terminal region of IL-3 mediate binding to the two different chains of the IL-3 receptor and emphasize the functional significance of the conserved Glu in the first helix of the IL-3, granulocyte-macrophage colony-stimulating factor, and IL-5 cytokine subfamily.

Amino Acid Sequence↗
PubMed1994

Dissociation of TNF-alpha cytotoxic and proinflammatory activities by p55 receptor- and p75 receptor-selective TNF-alpha mutants.

Human tumour necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine capable of killing mammalian tumour cells in vitro and in vivo, and of enhancing the proinflammatory activity of leucocytes and endothelium, the latter effects limiting its usage as an antitumour agent in humans. Using TNF-alpha mutants with a selective capacity to bind to the TNF p55 receptor (TNFR55) or to the p75 receptor (TNFR75) we show here that these two major activities of TNF-alpha can be dissociated. The TNFR55-selective mutants (R32W, E146K and R32W-S86T) which bind poorly to TNFR75 displayed similar potency to wild-type TNF in causing cytotoxicity of a human laryngeal carcinoma-derived cell line (HEp-2) and cytostasis in a human leukaemic cell line (U937). However, these TNFR55-selective mutants exhibited lower proinflammatory activity than wild-type TNF. Specifically, TNF-alpha's priming of human neutrophils for superoxide production and antibody-dependent cell-mediated cytotoxicity, platelet-activating factor synthesis and adhesion to endothelium were reduced by up to 170-fold. Activation of human endothelial cell functions represented by human umbilical venular endothelial cell (HUVEC) adhesiveness for neutrophils, E-selectin expression, neutrophil transmigration and IL-8 secretion were also reduced by up to 280-fold. On the other hand, D143F, a TNFR75-selective mutant tested either alone or in combination with TNFR55-selective mutants, did not stimulate these activities despite being able to cause cytokine production in TNFR75-transfected PC60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1994

T-cell functional regions of the human IL-3 proximal promoter.

The human interleukin-3 (IL-3) gene is expressed almost exclusively in activated T cells. Its expression is regulated at both the transcriptional and post-transcriptional level. We have previously shown that treatment of Jurkat T cells with phytohemaglutinin (PHA) and the phorbol ester, PMA, activated transcription initiation from the IL-3 gene. To define the regions of the gene required for transcription activation, we generated a series of reporter constructs containing different regions of the IL-3 gene 5' and 3' flanking sequences. Both positive and negative regulatory elements were identified in the proximal 5' flanking region of the IL-3 gene. The promoter region between -173 and -60 contained the strongest activating elements. The transcription factor AP-1 could bind to this positive activator region of the promoter. We also examined the function of the IL-3 CK-1/CK-2 elements that are present in many cytokine genes and found that they acted as a repressor of basal level expression when cloned upstream of a heterologous promoter but were also inducible by PMA/PHA.

Base Sequence↗
PubMed1994

The role of granulocyte-macrophage and granulocyte colony-stimulating factors in neutrophil transendothelial migration: comparison with interleukin-8.

Granulocyte-macrophage and granulocyte colony-stimulating factors (GM-CSF and G-CSF) have proinflammatory effects on mature neutrophils. Both factors have been reported to cause neutrophil chemotaxis and are produced by cells stimulated by inflammatory mediators, including endothelial cells. We therefore tested the hypothesis that these factors might mediate neutrophil transendothelial migration, either by forming a gradient across the endothelial monolayer or through the production of CSFs by activated endothelium. Studies of neutrophil migration across filters without endothelium showed that migration was promoted in the presence of a gradient of either CSF, but was equally promoted against the gradient; that is, the CSFs are chemokinetic but not chemotactic. The CSFs promoted migration of neutrophils across endothelial monolayers cultured on filters, but the magnitude of this effect was very small compared with that of a prototypic neutrophil chemoattractant, interleukin-8 (IL-8) (migration index [stimulated/unstimulated] 1.8-fold for GM-CSF, 10.8-fold for IL-8). Activation of endothelial monolayers by preincubation with tumor necrosis factor-alpha (TNF-alpha) increased neutrophil transmigration significantly; neutralizing antibodies to IL-8 inhibited this increase by 44%, whereas neutralizing anti-GM-CSF antibodies did not inhibit it. These data suggest little role for the CSFs in neutrophil diapedesis at inflammatory sites in vivo. Exposure of neutrophils to GM-CSF decreased their migration through TNF-activated monolayers, whereas G-CSF did not. This may have implications for the therapeutic administration of these factors.

Antibodies↗
PubMed1994

Characterization of a spontaneously transformed human endothelial cell line.

BACKGROUND: A line of cells was isolated from a focus observed in a human umbilical vein endothelial cell (HUVEC) culture, presumably the result of a spontaneous transformation event. EXPERIMENTAL DESIGN: The cell line was continuously cultured for 16 months, after which time, the proliferative rate, capacity to be cloned, and ability to be transfected was investigated. The cell line was analyzed for expression of endothelial cell markers, von Willebrand Factor, P selectin, and scavenger receptor. We also examined the tumor necrosis factor (TNF)-mediated upregulation of E-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1. We evaluated the levels of expression and types of TNF-alpha receptor expressed by the cell line, and the cell lines response to interleukin-4 and interferon-gamma. CD34 expression of this cell line and the ability of transforming growth factor-beta to inhibit the TNF-alpha induction of E-selectin was examined. The ability to support neutrophil adhesion and transmigration, and generate capillary-like tubes in vitro was assessed. Finally, the karyotype and tumourigenicity of this line was established. RESULTS: This cell line (C11STH) has a doubling time comparable to that of normal HUVECs and has a trisomy of chromosome 8 and 11. The cell line is capable of generating colonies at clonal density, and is transfectable with efficiencies comparable to normal HUVECs. C11STH expresses von Willebrand factor, P-selectin, and scavenger receptor to an extent similar to passaged HUVECs and can be induced to express E-selectin, VCAM, and ICAM with TNF-alpha. C11STH expresses both the p55 and p75 subunits of TNF-alpha receptor at levels similar to HUVECs. The ability of interleukin-4 to enhance the expression of VCAM and reduce the TNF-alpha-mediated expression of E-selectin is maintained in this cell line. C11STH cells are unable to induce class II major histocompatibility antigen in response to interferon-gamma. However, interferon-gamma is able to synergize with TNF to enhance the expression of E-selectin. C11STH cells do not express CD34 or show transforming growth factor-beta inhibition of TNF-alpha induced E-selectin expression, functions indicative of primary, or early passage HUVECs. The cell line retains the ability to support neutrophil adhesion and transmigration and can generate patent tubes when seeded onto complex basement membrane gels. However, the cell line no longer has the ability to generate capillary-like vessels when seeded onto collagen gels in the presence of phorbol 12-myristate 13-acetate. CONCLUSIONS: C11STH has retained many of the normal characteristics of endothelial cells, is able to proliferate at clonal density, and is easily transfectable. The line will provide a useful resource for endothelial cell biology. Its ability to make capillary-like tubes on basement membrane gels, but not on collagen will provide a powerful tool with which to further analyze the processes involved in angiogenesis, and will enable us to define the role of specific proteins in angiogenesis. Since C11STH shows tube competence on Matrigel, but is not tube competent on collagen, our studies suggest that capillary-tube formation on Matrigel and collagen occur via qualitatively different mechanisms. Thus, this cell line provides the opportunity to examine the signalling mechanisms required to generate capillary tube formation. These may include the involvement of matrix molecules, the production of proteases and inhibitors, gene regulation and kinases or phosphatases.

Cell Adhesion↗
PubMed1993

The receptor for interleukin 3 is selectively induced in human endothelial cells by tumor necrosis factor alpha and potentiates interleukin 8 secretion and neutrophil transmigration.

Interleukin (IL)-3 stimulates hemopoiesis in vitro. However, IL-3 is not normally found in bone marrow, raising doubts as to the in vivo role of IL-3. We have found that human umbilical vein endothelial cells (HUVEC) express functional high-affinity receptors for IL-3 after stimulation with tumor necrosis factor alpha (TNF-alpha), IL-1 beta, or lipopolysaccharide, and that this receptor is involved in inflammatory phenomena. TNF-alpha caused time- and dose-dependent up-regulation of mRNA for the IL-3 receptor alpha and beta chains, with maximal effects occurring 16-36 h after stimulation with TNF-alpha at 100 units/ml. Induction of mRNA correlated with protein expression on the cell surface as judged by monoclonal antibody staining and by the ability of HUVEC to specifically bind 125I-labeled IL-3. Scatchard analysis under optimal conditions of TNF-alpha stimulation revealed approximately 1500 IL-3 receptors per cell, which were of a high-affinity class (Kd = 500 pM) only. In contrast to a previous report, receptors for granulocyte-macrophage colony-stimulating factor could not be detected. IL-3 binding to TNF-alpha-activated HUVEC enhanced IL-8 production, E-selection expression, and neutrophil transmigration. The selective induction of a functional IL-3 receptor on endothelial cells suggests that, beyond hemopoiesis, IL-3 may have an important role in chronic inflammation and in allergic diseases.

Cell Adhesion Molecules↗
PubMed1993

A cytokine receptor gene cluster in the X-Y pseudoautosomal region?

The receptors for interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are heterodimers comprised of ligand specific alpha chains and a common beta chain. The genes encoding the IL-5 receptor alpha chain and the common beta chain reside on chromosome 3 and 22 respectively, while the GM-CSF receptor alpha chain gene (CSF2RA) has been mapped to the pseudoautosomal region (PAR) of the sex chromosomes, which is a 2.6-Mb stretch of homologous sequence at the tips of the short arms within which a single obligatory recombination occurs during male meiosis. We have mapped the gene encoding the IL-3 receptor alpha chain (IL3RA) to the sex chromosomes by polymerase chain reaction (PCR) analysis of human-mouse or human-chinese hamster cell hybrids, and to Yp13.3 and Xp22.3 using fluorescence in situ hybridization. To explore the possibility that IL3RA is located within the pseudoautosomal region we screened the Centre d'Etude du Polymorphisme Humain (CEPH) pedigrees for an informative-restriction fragment-length polymorphism (RFLP) that showed male meiotic recombination. Two informative CEPH pedigrees were identified that displayed this phenomenon, confirming the psuedoautosomal location of IL3RA. Using long-range restriction mapping we have found that IL3RA maps to the same 190-kb restriction fragment as CSF2RA, suggesting that a cytokine receptor gene cluster may reside in the PAR.

Base Sequence↗
PubMed1993

Transforming growth factor-beta inhibits E-selectin expression on human endothelial cells.

Transforming growth factor-beta (TGF-beta), a pleiotropic cytokine that is elaborated in the active form upon co-culture of endothelial cells and pericytes or smooth muscle cells, has been shown to decrease the adhesiveness of endothelial cells for neutrophils, lymphocytes, and tumor cells. The mechanism whereby TGF-beta inhibits the adhesiveness of human endothelial cells was investigated. TGF-beta inhibited the basal E-selectin (formerly ELAM-1) expression by 55 +/- 7% and TNF-stimulated expression by 57 +/- 4%. Similar decreases of IL-1-stimulated expression were also seen. Peak inhibition was seen at TGF-beta doses between 0.2 and 2 ng/ml. Both TGF-beta 1 and -beta 2 were functional. The effectiveness of TGF-beta in inhibiting E-selectin expression was dependent on cell density and incubation time. TGF-beta also inhibited E-selectin mRNA levels in endothelial cells. TGF-beta had no effect on the expression of VCAM-1 and ICAM-1, but was additive with IL-4 in inhibiting the expression of E-selectin. The expression of E-selectin has been shown to mediate several aspects of the inflammatory response involving neutrophils and memory T lymphocytes. Perivascular TGF-beta appears to act as an inhibitor of the expression of the endothelium-specific selectin, E-selectin, and therefore of inflammatory responses involving neutrophils and (a subset of) lymphocytes.

Base Sequence↗
PubMed1993

The granulocyte-macrophage colony-stimulating factor/interleukin 3 locus is regulated by an inducible cyclosporin A-sensitive enhancer.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) are pleiotropic hemopoietic growth factors whose genes are closely linked and induced in T lymphocytes in a cyclosporin A (CsA)-sensitive fashion. Since we found that the human GM-CSF and IL-3 proximal promoters were not sufficient to account for the observed regulation of these genes, we mapped DNase I hypersensitive sites across the GM-CSF/IL-3 locus in the Jurkat human T-cell line to identify additional regulatory elements. We located an inducible DNase I hypersensitive site, 3 kb upstream of the GM-CSF gene, that functioned as a strong CsA-sensitive enhancer of both the GM-CSF and IL-3 promoters. Binding studies employing Jurkat cell nuclear extracts indicated that four sites within the enhancer associate with the inducible transcription factor AP1. Three of these AP1 elements lie within sequences that also associate with factors resembling the CsA-sensitive, T cell-specific transcription factor NFAT. We provide additional evidence suggesting that an AP1-like factor represents one of the components of NFAT. We propose that the intergenic enhancer described here is required for the correctly regulated activation of both GM-CSF and IL-3 gene expression in T cells and that it mediates the CsA sensitivity of the GM-CSF/IL-3 locus.

Base Sequence↗
PubMed1993

Regulation of in vitro capillary tube formation by anti-integrin antibodies.

Human endothelial cells are induced to form an anastomosing network of capillary tubes on a gel of collagen I in the presence of PMA. We show here that the addition of mAbs, AK7, or RMAC11 directed to the alpha chain of the major collagen receptor on endothelial cells, the integrin alpha 2 beta 1, enhance the number, length, and width of capillary tubes formed by endothelial cells derived from umbilical vein or neonatal foreskins. The anti-alpha 2 beta 1 antibodies maintained the endothelial cells in a rounded morphology and inhibited both their attachment to and proliferation on collagen but not on fibronectin, laminin, or gelatin matrices. Furthermore, RMAC11 promoted tube formation in collagen gels of increased density which in the absence of RMAC11 did not allow tube formation. Neither RMAC11 or AK7 enhanced capillary formation in the absence of PMA. Lumen structure and size were also altered by antibody RMAC11. In the absence of antibody the majority of lumina were formed intracellularly from single cells, but in the presence of RMAC11, multiple cells were involved and the lumen size was correspondingly increased. Endothelial cells were also induced to undergo capillary formation in fibrin gels after PMA stimulation. The addition of anti-alpha v beta 3 antibodies promoted tube formation in fibrin gels and inhibited EC adhesion to and proliferation on a fibrinogen matrix. The enhancement of capillary formation by the anti-integrin antibodies was matrix specific; that is, anti-alpha v beta 3 antibodies only enhanced tube formation on fibrin gels and not on collagen gels while anti-alpha v beta 1 antibodies only enhanced tubes on collagen and not on fibrin gels. Thus we postulate that changes in the adhesive nature of endothelial cells for their extracellular matrix can profoundly effect their function. Anti-integrin antibodies which inhibit cell-matrix interactions convert endothelial cells from a proliferative phenotype towards differentiation which results in enhanced capillary tube formation.

Animals↗
PubMed1993

A model for the interaction of the GM-CSF, IL-3 and IL-5 receptors with their ligands.

The high affinity receptors for GM-CSF, IL-3 and IL-5 are heterodimers consisting of a ligand-specific alpha chain and a common beta chain. These proteins are members of a family of proteins known as the "cytokine receptor family" which is characterized by the presence of a 200-residue ligand-binding module. The GM-CSF, IL-3 and IL-5 receptor alpha chains constitute a distinct subgroup and share features not found in other members of the cytokine receptor family, features which we propose to be important for their interaction with the common beta chain and for their binding of the structurally-related ligands. The growth hormone receptor is a well-characterized member of the cytokine receptor family. Based on the structure of the complex between growth hormone and its receptor, we have proposed sites of contact between the GM-CSF, IL-3 and IL-5 receptors and their cognate ligands.

Amino Acid Sequence↗
PubMed1993

Chemotactic desensitization of neutrophils demonstrates interleukin-8 (IL-8)-dependent and IL-8-independent mechanisms of transmigration through cytokine-activated endothelium.

We have recently shown that an exogenous gradient of interleukin-8 (IL-8) induces the transendothelial migration of neutrophils. Treatment of endothelium with the cytokines IL-1 or tumour necrosis factor (TNF) also causes neutrophil transmigration, and recent evidence suggests that this may be due to endogenous IL-8 produced by the endothelium. We have used specific chemotactic desensitization of neutrophils to investigate the role of IL-8 in transmigration through cytokine-activated endothelium. Preincubation of neutrophils with IL-8 reduced their chemotactic transmigration response to an IL-8 gradient by 81%, demonstrating desensitization. Transmigration in response to cytokine-activated endothelium was inhibited by 104% after IL-8 preincubation, thus tending to support the role of IL-8. However, preincubation with another neutrophil chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (FMLP), which did not affect the IL-8, response, also inhibited transmigration, by 74%. This suggests that FMLP preincubation acts to inhibit a non-IL-8-dependent mechanism of transmigration through cytokine-activated endothelium. Chemotactic factor pretreatment of neutrophils did not reduce their adhesion to activated endothelium, but specifically blocked the transmigration step. We have therefore shown that chemotactic transmigration can be subjected to factor-specific desensitization, and have used this to provide evidence supporting a role for IL-8 in transmigration through cytokine-activated endothelium, as well as suggesting a further IL-8-independent mechanism. These data also provide a mechanism for the observed defect in accumulation of neutrophils at inflammatory sites when chemotactic factors are infused intravenously.

Cells, Cultured↗