Interleukin-8 and related chemotactic cytokines. The Giles Filley Lecture.
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Animals↗
Biomedical subjects
I Clark-Lewis
Publications and source records attributed to I Clark-Lewis.
Carbohydrate does not modulate the in vivo effects of injected interleukin-3.
Murine interleukin-3 (IL-3) has extensive N-linked glycosylation. Experiments were performed to determine whether the T cell-derived glycosylated IL-3 differs in its biological activity in vivo when compared with a chemically synthesized form of nonglycosylated IL-3. Groups of mice were treated by intravenous injection with identical units of IL-3 bioactivity as determined in vitro in a cell-proliferation assay. Mice that were treated with seven 5000-unit doses of either form of IL-3, given in 12-hour intervals, showed a small but significant increase in the frequency of mast cell precursor cells in the spleen and of IL-3-responsive colony-forming unit cells (CFU-C). There was no difference in potency of glycosylated and nonglycosylated IL-3. Induction, by IL-3, of histidine decarboxylase in bone marrow and spleen cells was used as a second measure for IL-3 bioactivity. Both IL-3 preparations showed good in vivo histidine decarboxylase inducing activity; however, T cell-derived glycosylated IL-3 was significantly more effective than synthetic IL-3 in inducing the enzyme histidine decarboxylase in bone marrow and in spleen cells. Pharmacokinetic studies showed that chemically synthesized IL-3 was cleared about twice as fast as the T cell-derived IL-3 and that there may be some tissue trapping of glycosylated IL-3. The shorter in vivo half-life of nonglycosylated IL-3 appears to have significant pharmacological consequences on the short-term effect of inducing histidine decarboxylase activity, but not on the effect of the long-term treatment of IL-3 on stimulating the increase of hematopoietic progenitor cells.
Animals↗
Binding to heparan sulfate or heparin enhances neutrophil responses to interleukin 8.
The interaction of interleukin 8 (IL-8) with heparin was studied by using synthetic IL-8 analogs with C- and N-terminal truncations. Elimination of the N-terminal region preceding the first cysteine, which constitutes the IL-8 receptor binding site, did not affect the affinity to heparin-Sepharose. Affinity, however, decreased with progressive truncation at the C terminus, and no binding was observed when the C-terminal alpha-helix was eliminated. The effect of heparin and other glycosaminoglycans on IL-8 activity was also tested. When IL-8 was applied together with heparan sulfate, neutrophil chemotaxis in vitro was enhanced up to 4-fold, and the stimulus-dependent increase in cytosolic free Ca2+ increased markedly in both rate and peak value. Heparin had a similar effect on the Ca2+ response but did not enhance chemotaxis. The glycosaminoglycans by themselves did not elicit neutrophil responses. Their enhancing effect was restricted to stimulation with IL-8 and was not observed when the unrelated chemoattractant fMet-Ile-Phe-Leu was used as the stimulus. Elastase released from stimulated neutrophils was inhibited by heparin, heparan sulfate, and, to a lesser extent, chondroitin sulfate B, confirming previous observations. Taken together, these results suggest that heparan sulfate, which is present on the endothelial cell surface and in the basement membrane, may have a dual function in diapedesis, promotion of IL-8-dependent transmigration of neutrophils, and protection of the tissue microenvironment from damage by lytic enzymes released from the migrating cells.
Amino Acid Sequence↗
The interleukin-8-related chemotactic cytokines GRO alpha, GRO beta, and GRO gamma activate human neutrophil and basophil leukocytes.
GRO alpha, a protein structurally related to interleukin-8 (IL-8) and originally described as a melanoma growth stimulatory factor, possesses potent neutrophil-stimulating activity. Recently, two closely related genes, gro beta and gro gamma, were identified. In the present work, the three GRO proteins were chemically synthesized, and their biological activities on human neutrophils and other leukocytes were compared. GRO alpha, GRO beta, and GRO gamma, like IL-8, induced chemotaxis, shape change, a rise in intracellular free calcium levels, exocytosis, and the respiratory burst in neutrophils. The GRO proteins were also active toward basophils as shown by chemotaxis and intracellular calcium concentration changes. The order of potency in neutrophils and basophils was IL-8 > GRO alpha > or = GRO gamma > GRO beta. Of the two IL-8 receptors expressed on human neutrophils, one binds GRO alpha with high and the other with low affinity. Competition binding experiments using radiolabeled IL-8 and GRO alpha revealed the same characteristics for GRO beta and GRO gamma. Similarly, cross-desensitization, as assessed by the stimulus-dependent changes in intracellular calcium concentration, indicated that all three GRO proteins interact with common receptors. From these results, it can be concluded that GRO alpha, GRO beta, and GRO gamma have the same pattern of activity toward human granulocytes and that the differences in amino acid sequence among these proteins have only minor effects on biological activity.
Amino Acid Sequence↗
Platelet factor 4 binds to interleukin 8 receptors and activates neutrophils when its N terminus is modified with Glu-Leu-Arg.
Amino acid deletion and mutagenesis experiments have indicated that the sequences Glu-Leu-Arg (ELR) preceding the first cysteine at the N terminus of interleukin 8 (IL-8) is required for receptor binding and neutrophil activation. Platelet factor 4 (PF4) is structurally related to IL-8 (35% sequence identity) but lacks the N-terminal ELR sequence and comparable effects on neutrophils. We introduced the ELR sequence at the N terminus of PF4 and found that the modified protein was a potent neutrophil activator and attractant. On the other hand, when the ELR sequence was introduced into the corresponding positions of two other proteins related to IL-8, gamma-interferon-inducible protein IP10 and monocyte chemoattractant protein 1, neither of them acquired neutrophil-activating properties, indicating that besides ELR additional structural determinants of IL-8 and PF4 are important for binding to IL-8 receptors. The conservation of these binding determinants suggests that PF4 may have evolved from a neutrophil activating protein.
Amino Acid Sequence↗
Interleukin-8 antagonists generated by N-terminal modification.
We have previously shown that the residues Glu4-Leu5-Arg6 (ELR) preceding the first cysteine at the N terminus of the 72-residue form of interleukin-8 (IL-8) are essential for receptor binding and neutrophil activation (Clark-Lewis, I., Schumacher, C., Baggiolini, M., and Moser, B. (1991) J. Biol. Chem. 266, 23128-23134). We have now synthesized a series of analogs of IL-8(4-72), the truncated form of IL-8 with the N-terminal sequence ELRC, as potential IL-8 antagonists. Among 26 analogs with deletions or amino acid replacements in the ELR region several inhibited IL-8 function. The most potent were IL-8(6-72), with Arg6 at the N terminus, and IL-8,AAR(7-72) with N-terminal Ala4-Ala5 instead of Glu4-Leu5. They inhibited IL-8 receptor binding, exocytosis (IC50 0.3 microM), as well as chemotaxis and the respiratory burst. Inhibition was restricted to responses elicited by IL-8, GRO alpha, or NAP-2, and no effect was observed when the unrelated agonists fMet-Leu-Phe or C5a were used as stimuli. These results demonstrate that selective antagonists that prevent or attenuate the action of IL-8 and its related chemotactic cytokines are obtained by modification of the ELR sequence at the N terminus.
Amino Acid Sequence↗
Molecular cloning of the cDNA and chromosomal localization of the gene for a putative seven-transmembrane segment (7-TMS) receptor isolated from human spleen.
A family of proinflammatory cytokines sharing several structural features has been described and includes, for example, interleukin-8, monocyte chemoattractant protein-1, and melanocyte growth stimulatory activity. Recently, the receptors for interleukin-8 have been isolated and found to belong to the seven-transmembrane domain class of G protein-coupled receptors. As other members of this cytokine family likely interact with similar receptors, the polymerase chain reaction was employed to isolate related receptors from human peripheral blood adherent cells. Degenerate oligonucleotide primers based on the rabbit interleukin-8 receptor sequence were used. The corresponding full-length cDNA was isolated from a human spleen cDNA library. The predicted protein sequence of this clone, designated pBE1.3, was 93% identical to that of a cDNA isolated from bovine locus coeruleus, which apparently encodes a neuropeptide Y receptor, and also shows similarity with the interleukin-8 receptor and the human cytomegalovirus US28 sequences. The gene, designated D2S201E was localized to human chromosome 2q21. By Northern blotting, transcripts hybridizing to this cDNA were present in a variety of tissues and cells, including those of hemopoietic origin.
Amino Acid Sequence↗
Structure-function relationships of lipoprotein lipase: mutation analysis and mutagenesis of the loop region.
The molecular models of two microbial lipases and human pancreatic lipase (PL) have suggested the existence of common structural motifs including a buried active site shielded by an amphipathic surface loop. In an effort to explore the role of residues comprising the loop of lipoprotein lipase (LPL), we have used site-directed mutagenesis to generate three new LPL variants. In variant LPLM1 we deleted 18 amino acids leaving a loop of only 4 residues which resulted in an LPL protein inactive against triolein substrates. In contrast, two other LPL variants with only partial deletions, involving the apical section of the loop [LPLM2 (-8 amino acids) and LPLM3 (-2 amino acids)] manifested normal lipolytic activity. These findings indicate a critical requirement for the maintenance of charge and periodicity in the proximal and distal segments of the LPL loop in normal catalytic function. This is further highlighted by the detection of a mutation in the proximal section of the loop in a patient with LPL deficiency at position 225 which results in a substitution of threonine for isoleucine. The intact catalytic activity of the partial deletion variants (LPLM2 and LPLM3) further suggests that the apical residues of the loop contribute minimally to the functional motifs of the active site. We support this postulate by showing that the conserved glycine in the apical turn section (G229) can be substituted by glutamine, lysine, proline, or threonine without significantly affecting catalytic activity.
Base Sequence↗
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↗
High- and low-affinity binding of GRO alpha and neutrophil-activating peptide 2 to interleukin 8 receptors on human neutrophils.
GRO alpha and neutrophil-activating peptide 2 (NAP-2), like their analog interleukin 8 (IL-8), are considered to be inflammatory mediators since they recruit and activate neutrophil leukocytes. After introduction of tyrosines by substitution for other residues at the C terminus, GRO alpha and NAP-2 were labeled with 125I and used for binding studies. A total of 60,000-90,000 receptors per neutrophil were found for either ligand. Of these 30-45% were of high affinity with a mean Kd value of 0.3 and 0.7 nM for GRO alpha and NAP-2, respectively, and 55-70% of low affinity (Kd = 30 nM). Two proteins of approximately 70 kDa and 44 kDa (p70 and p44) were specifically cross-linked with labeled GRO alpha, NAP-2, and IL-8. Unlabeled IL-8 fully inhibited this cross-linking and the binding of labeled GRO alpha or NAP-2 to the high-affinity sites on neutrophils or neutrophil membranes. Treatment of membranes with digitonin resulted in the preferential solubilization of a single receptor species, corresponding to p44, that bound GRO alpha and NAP-2 with low affinity (Kd = 30 nM) and IL-8 with high affinity (Kd = 0.4 nM). Exposure of neutrophil membranes to 100 microM guanosine 5'-[gamma-thio]triphosphate led to a 75-fold increase of the Kd in approximately 60% of the IL-8 receptors. High-affinity receptors for GRO alpha and NAP-2 were similarly affected. In contrast, guanosine 5'-[gamma-thio]triphosphate had no effect on the binding of IL-8 to p44 solubilized by digitonin. These results demonstrate that human neutrophils bear two classes of receptors for GRO alpha, NAP-2, and IL-8 (p70 and p44) that may differ in their mode of interaction with GTP regulatory proteins.
Cell Membrane↗
Tyrosine phosphorylation of receptor beta subunits and common substrates in response to interleukin-3 and granulocyte-macrophage colony-stimulating factor.
The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) consist of two polypeptides each belonging to a new class of molecules referred to as the hemopoietin receptor family. When expressed alone, receptor polypeptides of this family often bind their respective factors with lower affinity than the receptors identified in whole cells. Despite the lack of structural evidence for any enzymatic activity of the receptor polypeptides, both IL-3 and GM-CSF stimulate tyrosine phosphorylation of multiple intracellular substrates. We investigated IL-3 and GM-CSF receptor structure and signaling in a myeloid cell line, FDC-P1, which is dependent on either IL-3 or GM-CSF for growth. Antiphosphotyrosine antibodies were used to immunoprecipitate tyrosine-phosphorylated proteins from 32P-labeled cells or to probe immunoblots. Both IL-3 and GM-CSF stimulated the phosphorylation of a similar pattern of polypeptides on tyrosine. One tyrosine phosphorylated polypeptide migrated with M(r) = 135,000 and increased to 150,000 over a period of 10 min following stimulation of cells with IL-3 or GM-CSF. The M(r) = 135,000-150,000 polypeptide phosphorylated in response to IL-3 was shown to be primarily the Aic-2A polypeptide, the low affinity IL-3 receptor. Phosphatase treatment showed that the dramatic IL-3-induced shift in apparent molecular weight from M(r) = 125,000 in unstimulated cells was entirely due to phosphorylation. The closely related receptor, Aic-2B, was also tyrosine phosphorylated in response to IL-3, although to a lesser extent than Aic-2A. Treatment with GM-CSF resulted in tyrosine phosphorylation of the Aic-2B polypeptide exclusively. It was intriguing that GM-CSF treatment did affect the mobility of the Aic-2A polypeptide on polyacrylamide gels. Together, these results suggest that the Aic-2A polypeptide is part of the IL-3 receptor complex, but not the GM-CSF receptor. In contrast, the Aic-2B polypeptide is a component of the GM-CSF receptor, but it can also be utilized in an IL-3 receptor.
Animals↗
Interleukin-8, a chemotactic and inflammatory cytokine.
Interleukin-8 (IL-8) belongs to a family of small, structurally related cytokines similar to platelet factor 4. It is produced by phagocytes and mesenchymal cells exposed to inflammatory stimuli (e.g., interleukin-1 or tumor necrosis factor) and activates neutrophils inducing chemotaxis, exocytosis and the respiratory burst. In vivo, IL-8 elicits a massive neutrophil accumulation at the site of injection. Five neutrophil-activating cytokines similar to IL-8 in structure and function have been identified recently. IL-8 and the related cytokines are produced in several tissues upon infection, inflammation, ischemia, trauma etc., and are thought to be the main cause of local neutrophil accumulation.
Amino Acid Sequence↗
Structure-function analysis of casein kinase 2 with synthetic peptides and anti-peptide antibodies.
Casein kinase 2 (CK2) is a ubiquitous, multifunctional protein-seryl/threonyl kinase that has been implicated in cellular regulation. Synthetic peptides were patterned after three highly conserved regions in CK2: the N terminus (CK2-NT); the lysine-rich, kinase subdomain III segment (CK2-III) (nomenclature of Hanks et al. (Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241, 42-52)); and a 10-residue segment located near kinase subdomain X that is shared between CK2 and p34cdc2 (CK2/cdc2). The CK2-III and CK2/cdc2 peptides markedly stimulated the autophosphorylation of the alpha- and alpha'-subunits of purified CK2 from sea star oocytes, and they elicited up to 2-fold increases in its casein or phosvitin phosphotransferase activity. These peptides completely reversed nearly total inhibition of CK2 phosphotransferase activity toward itself, casein, and phosvitin by either heparin or poly(Glu,Tyr; 4:1), whereas CK2-NT was ineffective. Elution of CK2 from heparin-agarose with the CK2-III peptide indicated that this region of CK2 might mediate heparin binding to CK2. Affinity-purified rabbit polyclonal antibodies developed against both CK2-III and CK2/cdc2, but not CK2-NT, also produced up to 1.8-fold enhancements of the casein and phosvitin phosphotransferase activities of purified CK2. All three of the antipeptide antibody preparations immunoreacted with the alpha- and alpha'-subunits of CK2 on Western blots. These studies indicate that kinase subdomains III and X are involved in the modulation of CK2 phosphotransferase activity.
Amino Acid Sequence↗
Functional role of heme ligation in cytochrome c. Effects of replacement of methionine 80 with natural and non-natural residues by semisynthesis.
The nature of the axial ligation to heme iron has been suggested to be the major determinant of the oxidation-reduction potential of a particular cytochrome, but natural cytochromes that vary significantly in E'm invariably differ from one another in many ways. We proposed to clarify this issue by engineering many different ligation patterns within the same basic molecule, mitochondrial cytochrome c. Since many of the potentially informative substitutions require non-coded amino acids, semisynthesis was the approach we chose, and solid-phase peptide synthesis was used to make a set of nin 39-residue peptides that have been incorporated by autocatalytic fragment religation into the structure of horse cytochrome c. An additional two analogues modified at this position were made by chemical modification of the whole protein. As well as looking at the effect on reduction potential, we examined the effect of varying the ligand sphere on the efficiency of the autocatalytic fragment religation reaction, on the conformation of cytochrome c, on its spectroscopic properties, and in promoting electron transfer between heme c and other redox centers. Substitute residues were chosen to put sulfur, selenium, oxygen, and nitrogen, or even no ligating atom at all in the place of methionine sulfur. We found both subtle and dramatic alterations in spectral properties, which were informative about changes in internal structure and stability brought about by the modifications and which may be useful in identifying novel natural ligation patterns. An unexpected finding was that alanine 80 cytochrome c acquires a hemoglobin-like spectrum, and binds O2 most effectively. Reduction potential changes of greater than 300 mV with nitrogen, greater than 400 mV with oxygen, and greater than 300 mV with thiol sulfur ligation were observed, confirming that variation of the ligand sphere is indeed the most effective way in which the protein coat may modulate the potential of the redox center it encloses. Finally, we obtained more evidence that this axial ligand plays an active role in electron transfer and discovered that histidine could be even more effective in this role.
Amino Acids↗
Purification and initial characterization of the lymphocyte-specific protein-tyrosyl kinase p56lck from a baculovirus expression system.
A baculovirus expression system has been used to express large quantities of the lymphocyte-specific protein-tyrosyl kinase p56lck. A series of chromatographic steps, including the novel application of metalchelate affinity chromatography for protein kinase purification, were employed to obtain p56lck in a highly active form. Recombinant p56lck was purified to apparent homogeneity as determined by polyacrylamide gel electrophoretic analyses and was found to migrate in SDS gels as two related species, both with apparent molecular masses close to 56 kDa. p56lck phosphorylated all assayed substrates exclusively on tyrosyl residues, and underwent autophosphorylation at one principal site, also on a tyrosyl residue. p56lck displayed a high affinity for a synthetic peptide corresponding to the cytoplasmic domain (residues 52-164) of the T-cell receptor zeta-chain (TCR-zeta) (Km approximately 6.5 microM) but a low affinity for a peptide corresponding to its own autophosphorylation site (Km approximately 900 microM). p56lck was also found to be highly active for a purified protein-tyrosyl kinase (Vmax greater than 400 pmol.min-1.micrograms-1 using the TCR-zeta (52-164) as a substrate). A variety of agents were tested for their ability to inhibit p56lck, with zinc ions (I50 approximately 1.7 mM) and staurosporine (I50 approximately 500 nM) proving the most potent.
Animals↗
IP-10, a gamma-interferon-inducible protein related to interleukin-8, lacks neutrophil activating properties.
IP-10, a small, gamma-interferon-inducible protein with structural homology to interleukin-8 (IL-8), was prepared by automated chemical synthesis and compared with synthetic IL-8, GRO alpha and neutrophil-activating peptide 2 (NAP-2) for neutrophil stimulating activity. The following functions were tested: cytosolic free calcium changes, chemotaxis in vitro, respiratory burst, exocytosis of azurophil and specific granules from cytochalasin B-pretreated cells, and competition for IL-8 receptor binding. At 1, 10, 100 and 1000 nM, IP-10 was inactive in all assays, in contrast to the reference peptides which exhibited the expected neutrophil stimulating effects. In addition, IP-10 did not induce neutrophil accumulation after intradermal injection in rats, and did not act as IL-8 antagonist.
Calcium↗
Uptake of basic amino acids and peptides into liposomes in response to transmembrane pH gradients.
The uptake of derivatives of lysine and a pentapeptide (ala-met-leu-trp-ala) into large unilamellar vesicle (LUV) systems in response to transmembrane pH gradients has been examined. In these derivatives, the C-terminal carboxyl functions have been converted to methyl esters or amides. It is shown that the presence of a pH gradient (interior acidic) results in the rapid and efficient accumulation of these weak base amino acid and peptide derivatives into LUVs in a manner consistent with permeation of the neutral (deprotonated) form. It is suggested that this property may have general implications for mechanisms of transbilayer translocation of peptides, such as signal sequences, which exhibit weak base characteristics.
Amino Acid Sequence↗
Two polypeptides identified by interleukin 3 cross-linking represent distinct components of the interleukin 3 receptor.
Two receptor polypeptides have been identified in several studies by using cross-linking with interleukin 3 (IL-3). It has been suggested that proteolytic degradation of the larger polypeptide yields the lower molecular weight fragment, but there is little proof that this is the case. We have used several different approaches to characterize the polypeptides cross-linked in R6X or FDC-P1 cells. Several bifunctional cross-linkers of various sizes were tested to determine their effectiveness in cross-linking IL-3 to its receptor. The longer cross-linker gave the highest proportion of labeling of the low molecular weight band. Incubation in the absence of protease inhibitors caused a decrease in labeling of both cross-linked polypeptides, but no indication of a significant increase in the lower molecular weight band. Direct comparison of the two cross-linked polypeptides by V8 protease mapping showed no common peptides that might be expected if they were related molecules, except those derived from the iodinated IL-3. Digestion with N-glycanase resulted in a decrease in apparent molecular weight of 5000 in the larger polypeptide but a decrease of 15,000 in the smaller polypeptide. These results suggest that the 70-kd polypeptide identified by cross-linking of IL-3 represents a second binding chain of the receptor. By analogy with some of the other hemopoietin receptors, the 70- and 125-kd polypeptides may form a complex necessary for high affinity binding.
Amidohydrolases↗