Enhanced expression of eotaxin and CCR3 in atopic dermatitis.

Chemokines are thought to play an important part in the development of inflammation in atopic dermatitis. Eotaxin, a CC chemokine, is a potent chemoattractant and activator of human eosinophils, basophils and Th2 lymphocytes which acts via the chemokine receptor CCR3. We studied the expression of eotaxin and CCR3, as well as MCP-3, MIP-1alpha and interleukin-8, in atopic dermatitis and normal skin by immunohistochemistry and nested reverse transcriptase-polymerase chain reaction. Skin biopsy specimens were obtained from nonlesional and lesional skin of patients with atopic dermatitis and of nonatopic controls. Immunoreactivity and transcripts of eotaxin and CCR3 were significantly increased in lesional skin from atopic dermatitis, but not in nonatopic controls. In nonlesional atopic dermatitis samples CCR3 expression was also significantly increased at the mRNA and protein level, whereas eotaxin was increased at the mRNA level only. No significant difference in the expression of MCP-3, MIP-1alpha, and interleukin-8 was observed between skin samples from atopic dermatitis and nonatopic controls. The enhanced local production of eotaxin may lead to the recruitment of eosinophils and T lymphocytes, which both express CCR3 and contribute to the initiation and maintenance of inflammation.

Disulfide bridges in interleukin-8 probed using non-natural disulfide analogues: dissociation of roles in structure from function.

The structural and functional roles of the two disulfide bridges in interleukin-8 (IL-8) were addressed using IL-8 analogues with covalently modified disulfide bridges. The analogues were prepared using chemical synthesis by replacement of a cysteine for either homocysteine, penicillamine, or selenocysteine and on folding resulted in a covalently modified disulfide. Deletion of either of the two disulfide bridges by replacement of either cysteine pair with alanine resulted in loss of both structure and function. In contrast, all of the analogues with modified disulfide bridges had native tertiary fold as determined by nuclear magnetic resonance spectroscopic methods. Their structural similarity provided a rational basis for assessing the functional effects of the changes to the disulfide. Modification to the disulfide bridge between cysteines 9 and 50 had only a modest effect on IL-8 function. In contrast, alterations to the 7-34 disulfide bridge resulted in a dramatic reduction in biological potency. Thus, although both disulfide bridges are required for maintenance of the native tertiary fold, their role in determining IL-8 activity is distinct. We propose that 7-34 disulfide has a direct role in determining receptor binding and activation, whereas the 9-50 was not directly involved. The synthesis of non-natural disulfide analogues is a novel general approach to structure-activity relationships of disulfide bridges. The demonstration that the participation of disulfide bridges in function can be dissociated from their effects on the stability of the tertiary structure suggests that this method will lead to increased understanding of the roles of disulfide bridges in proteins.

N-terminal peptides of stromal cell-derived factor-1 with CXC chemokine receptor 4 agonist and antagonist activities.

Peptides corresponding to the N-terminal 9 residues of stromal cell-derived factor-1 (SDF-1) have SDF-1 activity. SDF-1, 1-8, 1-9, 1-9 dimer, and 1-17 induced intracellular calcium and chemotaxis in T lymphocytes and CEM cells and bound to CXC chemokine receptor 4 (CXCR4). The peptides had similar activities to SDF-1 but were less potent. Whereas native SDF-1 had half-maximal chemoattractant activity at 5 nM, the 1-9 dimer required 500 nM and was therefore 100-fold less potent. The 1-17 and a 1-9 monomer analog were 4- and 36-fold, respectively, less potent than the 1-9 dimer. Both the chemotactic and calcium response of the 1-9 dimer was inhibited by an antibody to CXCR4. The basis for the enhanced activity of the dimer form of SDF-1, 1-9 is uncertain, but it could involve an additional fortuitous binding site on the 1-9 peptide in addition to the normal SDF-1, 1-9 site. A 1-9 analog, 1-9[P2G] dimer, was found to be a CXCR4 antagonist. Overall this study shows that the N-terminal peptides are CXCR4 agonists or antagonists, and these could be leads for high affinity ligands.

Granulocyte chemotactic protein 2 acts via both IL-8 receptors, CXCR1 and CXCR2.

Interleukin-8 (IL-8) acts on human neutrophils via two receptors, CXCR1 and CXCR2. It shares CXCR2 with all neutrophil-activating chemokines, which like IL-8 have a conserved Glu-Leu-Arg (ELR) N-terminal motif, but is generally considered to be the only relevant agonist for CXCR1. IL-8 has a basic residue at the sixth position after the second cysteine, which was suggested to contribute to CXCR1 specificity. Among the other ELR chemokines, only granulocyte chemotactic protein 2 (GCP-2) has such a basic determinant. Using Jurkat cells that stably express either CXCR1 or CXCR2, we studied receptor activation by IL-8, GCP-2 epithelial neutrophil-activating protein 2 (ENA-78) (which shares 77% identical amino acids with GCP-2) and growth-regulated oncogene alpha (GRO alpha). At 10 nM and higher concentrations, GCP-2 and IL-8 induced significant activation of CXCR1-expressing cells, but no activity was found with GRO alpha and ENA-78. As expected, however, all four chemokines had similar activities on CXCR2-expressing cells. A variant of GCP-2 in which the basic residue, Arg20, was replaced by a glycine was synthesized. This derivative was ineffective on CXCR1, but was as active as wild-type GCP-2 in CXCR2-expressing cells. GCP-2 displaced radiolabeled IL-8 from both receptors with low affinity, and in this respect resembled ENA-78 and GRO alpha. Our data show that GCP-2 acts via both IL-8 receptors and thus appears to be functionally more similar to IL-8 than to the other ELR chemokines. Activation of CXCR1 appears to depend significantly on the presence of a basic binding determinant close to the second cysteine.

Interleukin-8 expression in the urine after bacillus Calmette-Guerin therapy: a potential prognostic factor of tumor recurrence and progression.

PURPOSE: We assessed whether interleukin-8 (IL-8), one of the first cytokines expressed in the urine after bacillus Calmette-Guerin (BCG) therapy for superficial urothelial tumors, may serve as a prognostic factor for treatment response. MATERIALS AND METHODS: Of 20 patients with superficial urothelial cancer of the urinary tract treated with BCG 13 had superficial bladder cancer and 7 received BCG perfusion of the upper urinary tract due to inoperability, solitary kidney, renal insufficiency or bilateral disease. After intravesical instillation of 120 mg. BCG or after 2-hour perfusion of the upper urinary tract with 360 mg. BCG urine was collected at 6-hour intervals. IL-8 was determined by solid phase double ligand enzyme-linked immunosorbent assay. RESULTS: IL-8 was stable in the urine for more than 48 hours. At a median followup of 36.5 months treatment failure occurred in 10 of the 20 patients, including 3 with recurrence, 6 with progressive disease and 1 with extensive carcinoma in situ. IL-8 excretion was more than 4,000 ng. in the first 6 hours after BCG therapy in all 10 patients who remained disease-free. In 9 of the 10 patients with recurrent disease IL-8 excretion was less than 4,000 ng. in the first 6 hours after BCG therapy. Patients secreting less than 4,000 ng. IL-8 into the urine during the first 6 hours after BCG therapy had a significantly higher risk of tumor recurrence and progression (p <0.0002). CONCLUSIONS: Due to its stability and kinetics IL-8 determined in urine collected during the first 6 hours after BCG therapy may prove to be a prognostic factor for tumor recurrence and progression after BCG therapy.

In vitro modulation of cytokine, cytokine inhibitor, and prostaglandin E release from blood mononuclear cells and synovial fibroblasts by antirheumatic drugs.

OBJECTIVE: To assess the effect of various antirheumatic drugs on cytokine, cytokine inhibitor, and prostaglandin E (PGE) production by normal blood mononuclear cells (MNC) and rheumatoid arthritis (RA) synovial fibroblasts in vitro. METHODS: MNC from healthy donors and RA synovial fibroblasts were preincubated with or without prostaglandin E2 (PGE2), indomethacin, dexamethasone, gold sodium thiomalate (GSTM), methotrexate (MTX), and cyclosporin A (CyA), and then cultured in the absence or presence of interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) for 48 h. We characterized cytokines such as IL-1 beta, IL-8, monocyte chemoattractant protein-1 (MCP-1), and cytokine inhibitors such as IL-1 receptor antagonist (IL-1ra) and soluble TNF receptors (sTNFR p55 + p75) as well as PGE in the cell-free culture supernatants. RESULTS: In MNC and synovial fibroblast cultures dexamethasone, GSTM, and PGE2 most markedly downregulated spontaneous and/or cytokine stimulated production of IL-1 beta, IL-14a, IL-8, and MCP-1, whereas sTNFR shedding was not affected. In contrast, MTX and CyA had only marginal or no effects on mediator release, whereas indomethacin inhibited only PGE production. CONCLUSION: Among several antirheumatic drugs examined, dexamethasone and GSTM exhibited the most potent inhibitory effects on inflammatory cytokine and cytokine inhibitor production by blood mononuclear cells and synovial fibroblasts. These drugs may exert their antiinflammatory actions by unspecific suppression of monocyte and fibroblast secretory function.

Chemokine antagonists that discriminate between interleukin-8 receptors. Selective blockers of CXCR2.

Human neutrophils express two interleukin (IL)-8 receptors, CXC chemokine receptor (CXCR) 1 and CXCR2. IL-8 with changes to the NH2-terminal ELR motif can block IL-8-induced neutrophil functions (Moser, B., Dewald, B., Barella, L., Schumacher, C., Baggiolini, M., and Clark-Lewis, I. (1993) J. Biol. Chem. 268, 7125-7128). We have now examined the effect of NH2-terminally modified analogs of IL-8, GROalpha, and PF4 on CXCR1 and CXCR2 independently. Using stable Jurkat transfectants expressing either CXCR1 or CXCR2, it was shown that analogs derived from IL-8 bound both IL-8 receptors with similar affinity and could block IL-8-induced Ca2+ mobilization. By contrast, analogs of GROalpha and PF4, (R)GROalpha and (R)PF4, bound only CXCR2 with high affinity and blocked Ca2+ mobilization induced only via CXCR2. The differential effect on CXCR1 and CXCR2 was also demonstrated in studies with isolated neutrophils. Thus (R)GROalpha and (R)PF4 inhibited only the GROalpha but not the IL-8-stimulated elastase release, and these two analogs had no effect on IL-8-elicited superoxide generation, a response that is mediated by CXCR1 but not by CXCR2. These results show that CXCR2 selective receptor antagonists can be generated based upon the secondary binding determinants of GROalpha and PF4. They also highlight the primary importance of CXCR1 in chemokine-mediated release of granule enzymes and superoxide generation. The selective antagonists described may be used in future studies on IL-8 receptor signaling to define distinct steps leading to various functional responses induced in neutrophils via CXCR1 and CXCR2.

Properdin, a positive regulator of complement activation, is released from secondary granules of stimulated peripheral blood neutrophils.

Properdin is an important regulatory constituent of the complement system. In contrast to most other components of complement, biosynthesis of properdin is restricted to a few cell types only, i.e., monocytes/macrophages and peripheral blood T cells. This report demonstrates the presence of properdin mRNA in peripheral blood granulocytes and shows that properdin is stored in the granules of human neutrophils and secreted upon stimulation with TNF-alpha, C5a, IL-8, or FMLP. Subcellular fractionation using Percoll density gradients and Western blot analyses revealed that the bulk of properdin is contained in the secondary granules. Moreover, flow cytometric analyses indicated that properdin is present on the surface of neutrophils. In contrast to alternative pathway components, components of the classical pathway of complement activation, such as C2 and C4, were not detected. Our findings suggest that neutrophils can actively stabilize and amplify the alternative activation pathway of complement by secretion of properdin as part of the innate defense against microorganisms.

Neutrophil-activating peptide-2 and melanoma growth-stimulatory activity are functional as monomers for neutrophil activation.

Neutrophil-activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA) are members of the chemokine family of inflammatory proteins. The structures of NAP-2, determined by x-ray crystallography, and MGSA, elucidated by NMR spectroscopy, revealed a tetramer and dimer, respectively. In order to address the relevance of multimeric species to their activities on neutrophils, analogs of NAP-2 and MGSA were synthesized in which the backbone amide proton of Leu-22 in NAP-2, and Val-26 in MGSA, was substituted with the bulky methyl group (NH --> NCH3). These analogs were shown to be monomeric by sedimentation equilibrium ultracentrifugation studies and were similar to the corresponding native protein in assays for neutrophil elastase release and Ca2+ mobilization from IL-8R1 and IL-8R2 transformed cells. Sedimentation equilibrium studies of the native NAP-2 and MGSA were also carried out to address the association behavior. For NAP-2, there was no evidence for the tetramer, but an equilibrium between monomers and dimers and the dissociation constant was calculated to be 50-100 microM. Similarly, MGSA showed a monomer-dimer equilibrium with a Kd of approximately 5 microM. The data from the monomeric analogs and also the calculation of dissociation constants indicate that NAP-2 and MGSA have a tendency to associate above the concentrations required for maximal activity or for receptor activation, but at functional concentrations they are predominantly monomers.

Human chemokines: an update.

Interleukin 8, the first chemokine to be characterized, was discovered nearly ten years ago. Today, more than 30 human chemokines are known. They are often upregulated in inflammation and act mainly on leukocytes inducing migration and release responses. The present review deals largely with the new developments of the last three years. Several structural studies have shown that most chemokines form dimers. The dimers, however, dissociate upon dilution, and the monomers constitute the biologically active form. Chemokine activities are mediated by seven-transmembrane-domain, G protein coupled receptors, five of which were discovered in the past three years. The primary receptor-binding domain of all chemokines is near the NH2 terminus, and antagonists can be obtained by truncation or substitutions in this region. Major progress has been made in the understanding of chemokine actions on T lymphocytes that respond to several CC chemokines but also to IP10 and Mig, two CXC chemokines that selectively attract T cells via a novel receptor. Effects of chemokines on angiogenesis and tumor growth have been reported, but the data are still contradictory and the mechanisms unknown. Of considerable interest is the recent discovery that some chemokines function as HIV-suppressive factors by interacting with chemokine receptors which, together with CD4, were recognized as the binding sites for HIV-1.

1H NMR evidence that Glu-38 interacts with the N-terminal functional domain in interleukin-8.

In order to assess the importance of the buried Glu-38 observed in the structure of interleukin-8, an analog in which Glu-38 was replaced with Ala (E38A analog) was investigated by 1H NMR spectroscopy and neutrophil activation. Detailed analysis of the NMR NOESY data showed that the solution structure of the E38A analog is essentially the same as that for the native protein. Also, the neutrophil elastase activity of the E38A analog was similar to that of the native protein. However, the Gln-8 and Cys-9 amide proton chemical shifts, which are significantly downfield-shifted in the native protein, exhibit more 'normal' values. This observation indicates that in the native protein, Glu-38 side-chain carboxylate interacts with Gln-8 and Cys-9 amide protons. Although the N-terminal residues are critical for function, this interaction is not essential for neutrophil activation.

Opposite effects of interleukin-13 and interleukin-12 on the release of inflammatory cytokines, cytokine inhibitors and prostaglandin E from synovial fibroblasts and blood mononuclear cells.

We examined the effects of interleukin-12 (IL-12) and interleukin-13 (IL-13) on cytokine, cytokine inhibitor and prostaglandin E (PGE) release from synovial fibroblasts and blood mononuclear cells (MNC). In resting synovial fibroblasts, we found that IL-13 is an inhibitor of IL-8 and PGE release. A significant decrease of PGE synthesis caused by IL-13 was also observed in tumor necrosis factor (TNF)-alpha-stimulated synovial fibroblasts, whereas IL-12 had no regulatory effects on these cells. In resting and cytokine-stimulated MNC, IL-13 markedly inhibited IL-1 beta, IL-8 and monocyte chemoattractant protein-1 (MCP-1) release and potently stimulated interleukin-1 receptor antagonist (IL-1ra) synthesis. In contrast, IL-12 stimulated the production of IL-1 beta and MCP-1 in TNF-alpha-stimulated MNC and inhibited IL-1ra synthesis in cytokine-stimulated cells. These findings identify novel biological actions of IL-12 and IL-13 on connective tissue and on blood mononuclear cells which indicate their regulatory functions as enhancer and suppressor of inflammatory processes, respectively.

Interleukin 1 (IL-1) receptor antagonist, soluble tumor necrosis factor receptors, IL-1 beta, and IL-8--markers of remission in rheumatoid arthritis during treatment with methotrexate.

OBJECTIVE: To examine circulating levels of cytokines and cytokine inhibitors and their production by blood mononuclear cells (MNC) in patients with active rheumatoid arthritis (RA) before treatment with methotrexate (MTX) and inactive disease upon treatment as well as healthy control individuals. METHODS: Interleukin-1 receptor antagonist (IL-1ra), soluble tumor necrosis factor receptors p55 and p75 (sTNFr; p55 and p75), interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8), and monocyte chemoattractant protein 1 (MCP-1) were assessed by immunoassays in sera and MNC culture supernatants of 27 patients with RA with active disease before and 14 patients with inactive disease during MTX treatment, and 10 healthy controls. RESULTS: Levels of circulating IL-1ra, sTNFr p55 and p75 were higher in patients with active RA compared to those with inactive disease or controls. At the cellular level, resting MNC of patients with active RA released more IL-1 beta and IL-8, but less IL-1ra, and showed a lower ratio of IL-1ra:IL-1 beta than MNC of patients without inflammatory symptoms or healthy controls. In addition, unstimulated and in vitro lipopolysaccharide stimulated MNC cultures of patients with inactive RA released higher amounts of sTNFr p75 than MNC of patients with active RA. CONCLUSION: Circulating levels of IL-1ra and sTNFr as well as IL-1 beta, IL-8, and sTNFr p75 release from MNC and the ratio of IL-1ra:IL-1 beta production by these cells serve as markers to assess complete disease remission in patients with RA during MTX treatment.

Monocyte chemotactic protein 4 (MCP-4), a novel structural and functional analogue of MCP-3 and eotaxin.

A novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a baculovirus vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and cotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this cosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH2 terminus of an MCP is the biologically relevant species.

RANTES and MCP-3 antagonists bind multiple chemokine receptors.

Antagonists of multiple chemokines could be more effective than inhibitors of specific chemokines for controlling cell migration and inflammation. To attempt to identify such antagonists we characterized a number of truncated analogs of regulated on activation normal T cell expressed protein (RANTES), monocyte chemoattractant protein (MCP)-3, and MCP-1. On the basis of their ability to compete for binding of their parent chemokines, three analogs were selected for cross-reactivity studies: RANTES (9-68), MCP-3 (10-76), and MCP-1 (9-76). These analogs bound to THP-1 monocytic cells with dissociation constants that were within 4-6-fold of their native counterparts, but they did not promote detectable chemotaxis of THP-1 cells or enzyme release from purified human monocytes. The RANTES (9-68) analog competed for the binding and inhibited the activities of all three chemokines. In contrast, native RANTES was specific for RANTES binding sites. However, truncation of either MCP-1 or MCP-3 did not change their respective binding specificity. MCP-3 and MCP-3 (10-76) competed for binding of all three labeled chemokines. MCP-1 (9-76) competed strongly for binding of labeled MCP-1, but only weakly for the other two labeled ligands and inhibited the activities induced by MCP-1 and MCP-3 but not RANTES. Although RANTES (9-68) and MCP-3 (10-76) inhibited all three chemokines, the RANTES analog was significantly more potent for RANTES-induced activity. The results indicate that NH2-terminal residues partly determine the receptor specificity of RANTES, and deletions within this region permit binding to multiple chemokine receptors. The findings suggest the feasibility of design of high affinity multi-specific CC chemokine antagonists.

HCC-1, a novel chemokine from human plasma.

A novel CC chemokine, HCC-1, was isolated from the hemofiltrate of patients with chronic renal failure. HCC-1 has a relative molecular mass of 8,673 and consists of 74 amino acids including four cysteines linked to disulfide bonds. HCC-1 cDNA was cloned from human bone marrow and shown to code for the mature protein plus a putative 19-residue leader sequence. Mature HCC-1 has sequence identity of 46% with macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, and 29-37% with the other human CC chemokines. Unlike MIP-1 alpha and the other CC chemokines, HCC-1 is expressed constitutively in several normal tissues (spleen, liver, skeletal and heart muscle, gut, and bone marrow), and is present at high concentrations (1-80 nM) in plasma. HCC-1 has weak activities on human monocytes and acts via receptors that also recognize MIP-1 alpha. It induced intracellular Ca2+ changes and enzyme release, but no chemotaxis, at concentrations of 100-1,000 nM, and was inactive on T lymphocytes, neutrophils, and eosinophil leukocytes. In addition, HCC-1 enhanced the proliferation of CD34+ myeloid progenitor cells. It was as effective as MIP-1 alpha, but about 100-fold less potent.

Inhibition of the arachidonic acid pathway prevents induction of IL-8 mRNA by phorbol ester and changes the release of IL-8 from HL 60 cells: differential inhibition of induced expression of IL-8, TNF-alpha, IL-1 alpha, and IL-1 beta.

The promyelocytic HL 60 cell line can be used as an in vitro model system to study hematopoetic cell differentiation and inflammatory events. We studied the signal transduction pathway of induced interleukin (IL)-8 expression and compared it with those of tumor necrosis factor alpha (TNF-alpha), IL-1 alpha, and IL-1 beta. The differentiation of HL 60 cells to macrophage-like cells by PMA resulted in a rapid and marked induction of these inflammatory cytokines. The up-regulation occurred in the absence of ongoing protein synthesis, but cycloheximide-sensitive gene products modulated their induction kinetics. Staurosporine, a potent inhibitor of protein kinases, strongly inhibited their gene expression. Phosphorylation may not act directly on latent transcription factors, since bromophenacyl bromide, an inhibitor for the release of arachidonic acid from phorbol-12 myristate 13-acetate (PMA)-stimulated HL 60 cells, markedly depressed the induced mRNAs for IL-8, TNF-alpha, and IL-1 alpha and -beta. Similarly, 5,8,11,14 eicosatetraynoic acid (ETYA), another inhibitor of the arachidonic acid pathway, blocked the induction of transcripts for TNF-alpha, and both IL-1 genes in phorbol ester-stimulated HL 60 cells. In contrast, ETYA increased the induced IL-8 RNA levels and stimulated the release for IL-8. Also, ketoconazole, an inhibitor of 5-lipoxygenase and indomethacin, an inhibitor of cyclooxygenases did not block the induction of IL-8 mRNA. However, the release of IL-8 protein was regulated by indomethacin and ketoconazole. Our results indicate that arachidonic acid metabolites are mediators in the signal transduction pathway of IL-8 expression and that the involved second messengers are different from those which are important for the induction of TNF-alpha and IL-1 beta expression.

Methotrexate action in rheumatoid arthritis: stimulation of cytokine inhibitor and inhibition of chemokine production by peripheral blood mononuclear cells.

This open label study examines whether methotrexate (MTX) treatment modulates ex vivo synthesis of interleukin-1 receptor antagonist (IL-1ra), soluble tumour necrosis factor receptors (sTNFR p55 and p75), interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by peripheral blood mononuclear cells (PBMC) and whether changes reflect clinical response. Significant stimulation of IL-1ra and sTNFR p75 as well as inhibition of IL-8 production of PBMC were associated with clinical improvement observed in patients treated with MTX. When defining the characteristics of patients at study entry retrospectively in responders and non-responders, a significantly lower ratio of IL-1ra:IL-1 beta production before and its increase upon treatment was associated with clinical response in 13 patients compared to five patients not responding to MTX. In addition, clinical improvement was associated with decreased synthesis of IL-1 beta, TNF-alpha and IL-8 induced by bacterial lipopolysaccharide, IL-1 alpha and IL-1 beta in PBMC in vitro. These findings suggest that MTX therapy reverses the inflammatory type of rheumatoid arthritis (RA) blood mononuclear cells by stimulating cytokine inhibitor production while inhibiting inflammatory cytokine release at the same time. This may explain the powerful anti-inflammatory properties of low-dose MTX as observed in most RA patients. Pretreatment determination of the IL-1ra:IL-1 beta ratio in PBMC may be predictive with regard to a favourable therapeutic response and therefore may be useful for the selection of RA patients to be treated with MTX.

Structure-activity relationships of chemokines.

Structural analysis of chemokines has revealed that the alpha/beta structural-fold is highly conserved among both the CXC and CC chemokine classes. Although dimerization and aggregation is often observed, the chemokines function as monomers. The critical receptor binding regions are in the NH2-terminal 20 residues of the protein and are the least ordered in solution. The flexible NH2-terminal region is the most critical receptor binding site and a second site also exists in the loop that follows the two disulfides. The well-ordered regions are not directly involved in receptor binding but, along with the disulfides, they provide a scaffold that determines the conformation of the sites that are critical for receptor binding. These general requirements for function are common to all the chemokines. For the CC chemokines, receptor activation and receptor binding regions are separate within the 10 residue NH2-terminal region. This has allowed identification of high affinity analogs that do not activate the receptor and are potent antagonists.

Interleukin-10 differentially regulates cytokine inhibitor and chemokine release from blood mononuclear cells and fibroblasts.

In this study we have examined the effects of interleukin-10 (IL-10) on blood mononuclear cells (MNC) and on skin as well as on synovial fibroblasts. In unstimulated MNC, we found that IL-10 is a potent stimulator of interleukin-1 receptor antagonist (IL-1ra) and monocyte chemoattractant protein-1 (MCP-1) production and an inhibitor of IL-8 release. In cells exposed to IL-1 beta, it also moderately stimulated IL-1ra production and release of soluble tumor necrosis factor receptor p75 (sTNF-R p75) and inhibited IL-8 and MCP-1 production. In addition, we have evidence that the biological effects of IL-10 are not restricted to hematopoietic cells. IL-10 stimulated sTNF-R p55 dose-dependently and inhibited MCP-1 release from IL-1 beta-activated fibroblasts, whereas IL-8 production was not affected. Taken together, these findings identify novel biological actions of IL-10 on blood mononuclear and connective tissue cells which support its regulatory functions as a suppressor of inflammatory processes.