The predominance of beta (CC) chemokine transcripts in idiopathic inflammatory muscle diseases.

Cytokines and chemokines that upregulate major histocompatibility complex class I antigens, recruit lymphocytes, and enhance T-cell-mediated myotoxicity may be important in the pathogenesis of dermatomyositis and polymyositis. We searched for cytokine and chemokine transcripts in inflammatory muscle specimens from 14 newly diagnosed or treated patients. Control specimens from six patients without inflammatory muscle disease were analyzed for transcripts of interleukins-1 beta, -2, -4, -6, -10, and -15, and interferon-gamma, tumor necrosis factor-alpha, transforming growth factor-beta 1, macrophage inflammatory proteins-1 alpha and -1 beta (MIP-1 alpha, MIP-1 beta), and the chemokine "regulated on activation, normally T expressed and secreted" (RANTES). Surprisingly, the proinflammatory and lymphocyte cytokines were detected only sporadically in myositis muscle specimens, and their presence did not correlate with disease activity or treatment status of the patient. In contrast, MIP-1 alpha and MIP-1 beta were detected in 13 and 6 myositis biopsies, respectively, and RANTES, another beta (CC) chemokine, was detected in eight myositis biopsies. This study and other reports of low levels of acute-phase cytokines in myositis patients suggest that the proinflammatory cytokines do not play a major role in ongoing muscle damage. The CC chemokines studied here, in particular MIP-1 alpha, might contribute to ongoing muscle inflammation, and the pathogenesis of inflammation in myositis may follow a previously unrecognized pathway.

A motif in human histidyl-tRNA synthetase which is shared among several aminoacyl-tRNA synthetases is a coiled-coil that is essential for enzymatic activity and contains the major autoantigenic epitope.

In myositis, disease-specific autoantibodies may be directed against an aminoacyl-tRNA synthetase, usually histidyl-tRNA synthetase. To explore the basis for this phenomenon, we have made recombinant histidyl-tRNA synthetase in the baculovirus system. It was enzymatically active and recognized by human autoantibodies. A truncated protein lacking the first 60 amino acids was inactive as an antigen and as an enzyme. This region is within the first two exons, is predicted to have a coiled-coil configuration, and is found in some other synthetases but not in Escherichia coli or yeast histidyl-tRNA synthetase. Circular dichroism showed that the peptides from this region (amino acids 1-60 and 1-47) have the predicted high alpha-helical content, but smaller fragments (1-30, 14-45, and 31-60) do not. The peptides with a high alpha-helical content could inhibit autoantibodies almost completely, whereas the smaller peptides were unable to do so. The amino acid sequence of this coiled-coil domain in human histidyl-tRNA synthetase resembles the sequence of the extended this coiled-coil arm near the NH2 terminus of bacterial seryl-tRNA synthetase as well as similar regions in some eukaryotic aminoacyl-tRNA synthetases, raising the possibility that this domain serves a similar tRNA-stabilizing role and has been preserved from a common ancestor.

Long charge-rich alpha-helices in systemic autoantigens.

Autoimmune diseases are characterized by the presence of antibodies and T-cells targeting restricted sets of host proteins. This phenomenon may be due in part to greater non-specific immunogenicity for these proteins compared to others which are not autoantigenic. We used computer-based methods to analyze the sequenced human autoantigens for distinctive patterns of potential immunologic importance. Sequences longer than 27 residues predicted by these methods to form coiled-coil alpha-helices with a probability greater than 0.9 were detected in 40 of 109 (36.7%) of the known human autoantigens. These include many predominantly systemic disease-specific autoantigens not previously known to contain this structure. In comparison, 8.7% of human proteins in the Swissprot data base, and 1.1% of the proteins in the Brookhaven data base were found to contain such segments. These predicted coiled-coil alpha-helices are distinguished from most globular protein helices by greater length, higher charge content, and a heptad repeat multivalency. Coiled-coil segments correlate in part with known autoantibody epitopes and may contribute to autoantigenic potential. Systemic autoantigens generally are either basic or contain extended, multivalent, charge-rich segments such as coiled-coils.

Identification of macrophage cell-surface binding sites for cationized bovine serum albumin.

Autoimmune diseases are characterized by the presence of autoantibodies often restricted to host proteins exhibiting charge rich domains. Charged polypeptides elicit strong immune responses, and cationized bovine serum albumin and other cationic proteins are significantly more immunogenic than their less charged counterparts. These phenomena may involve enhanced protein uptake by macrophages, resulting in greater processing and presentation of antigenic peptide-MHC complexes to T-cells. We compared macrophage cell-surface binding and uptake of native and cationized bovine serum albumin. Specific binding of [125I]cationized bovine serum albumin to THP-1 macrophages in vitro was 11-16 fold greater than for native albumin. Half-maximal inhibition of [125I]cationized albumin binding was observed at 10-7M ligand. The specificity of [125I]cationized bovine serum albumin binding and uptake was further studied in terms of competitive inhibition of proteolysis by proteins of varying charge content. Cationized bovine serum albumin, but not native albumin, inhibited proteolysis of [125I]cBSA. Calf thymus histones also inhibited cBSA degradation. High concentration of myelin basic protein was moderately effective at blocking cBSA degradation, while myoglobin and beta lactalbumin showed no inhibition. These results indicate that specific cell-surface binding sites which occur on macrophages may mediate selective uptake of certain proteins with highly charged domains including some autoantigens.

Apolipoprotein A-I decreases neutrophil degranulation and superoxide production.

Neutrophils participate in the acute phase response and are often associated with tissue injury in a number of inflammatory disorders. The acute phase response is accompanied by alterations in the metabolism of apolipoprotein A-I and high density lipoprotein (HDL). Structural considerations led to studies investigating the effect of purified HDL and apolipoprotein A-I on neutrophil degranulation and superoxide production. Apolipoprotein A-I but not HDL inhibited IgG-induced neutrophil activation by about 60% as measured by degranulation and superoxide production. This suggests that the lipid-associating amphipathic helical domains of apolipoprotein A-I mediate this effect. In support of this was finding inhibitory effects with two synthetic model lipid-associating amphipathic helix peptide analogs. Apolipoprotein A-I, containing tandem repeating amphipathic helical domains, was approximately ten times more effective than the two peptide analogs and inhibited neutrophil activation at well below physiologic concentrations. Competitive binding studies indicate that resting neutrophils have approximately 190,000 (Kd = 1.7 x 10(-7)) binding sites per cell for apolipoprotein A-I, consistent with a ligand-receptor interaction. These observations suggest that apolipoprotein A-I may play an important role in regulating neutrophil function during the inflammatory response.

Very long charge runs in systemic lupus erythematosus-associated autoantigens.

Systemic lupus erythematosus and other chronic systemic autoimmune diseases are associated with circulating autoantibodies reactive with a limited set of mostly nuclear proteins. Using rigorous statistical methods we have identified segments of highly significant charge concentration in the majority of the characteristic nuclear and cytoplasmic autoantigens. Extremely long runs of charged residues, including some sequences of greater than 20 consecutive charged residues (purely acidic or mixed basic and acidic), occur in about a third of these proteins, whereas equivalent runs are found in less than 3% of other mammalian proteins. The other sequences have less extreme charge clusters, the type and location of which are often conserved between several otherwise nonsimilar antigens. We propose that supercharged surfaces render the targeted host proteins strongly immunogenic and that antinuclear antibody profiles might result from chronic exposure to intracellular contents, possibly in conjunction with crossreactive viral products. The limited number of potential systemic autoantigens may partly be due to the rarity of requisite charge properties.

Charge distributions and amphipathicity of receptor-binding alpha-helices.

Contacts between ligands and cell-surface receptors result in cellular activation. Defining principles which govern these important interactions are of interest and might facilitate pharmacologic intervention. We examined receptor-binding alpha-helical segments of polypeptide hormones and globular proteins for distinguishing amino acid content and distributions. There was a slight excess of basic residues in both sets of alpha-helices compared with a panel of control helices. Helical concentrations of charged residues were quantitated using the hydrophobic moment algorithm, adapted to obtain the vector sum of side chain charges. By this analysis we detected increased concentrations of the set of basic residues (arginine, lysine and histidine) on one side of the receptor-binding alpha-helices of the polypeptide hormones, and to a lesser extent the protein ligands. Comparable data were obtained for "lytic" venom peptides and calmodulin-regulated kinase segments. There was an even greater correlation between receptor-associating alpha-helical segments and large hydrophobic moments. Receptor-binding helical segments of polypeptide hormones, and to a lesser extent those of protein ligands, often are basic and amphipathic.

Identification of peptide hormones of the amphipathic helix class using the helical hydrophobic moment algorithm.

Eisenberg's helical hydrophobic moment (less than mu H greater than) algorithm was applied to the analysis of the primary structure of amphipathic alpha-helical peptide hormones and an optimal method for identifying other peptides of this class determined. We quantitate and compare known amphipathic helical peptide hormones with a second group of peptides with proven nonamphipathic properties and determine the best method of distinguishing between them. The respective means of the maximum 11 residue less than mu H greater than for the amphipathic helical and control peptides were 0.46 (+/-/-0.07) and 0.33 (0.07) (P + 0.004). To better reflect the amphipathic potential of the entire peptide, the percent of 11 residue segments in each peptide above a particular less than mu H greater than was plotted vs less than mu H greater than. The resulting curves are referred to as HM-C. The mean HM-C (of the two groups) was highly significantly different such that the HM-C method was superior to others in its ability to distinguish amphipathic from nonamphipathic peptides. Several potential new members of this structural class were identified using this approach. Molecular modeling of a portion of one of these, prolactin inhibitory factor, reveals a strongly amphipathic alpha helix at residues 4-21. This computer-based method may enable rapid identification of peptides of the amphipathic alpha-helix class.

Effect of eicosapentaenoic acid in asthma.

The role of arachidonic acid metabolites in the pathogenesis of airway inflammation and clinical asthma is currently unknown. The addition of eicosapentaenoic acid (EPA) to the diet of humans has been shown to generate metabolites that are less potent than their arachidonic acid counterparts. The substitution of EPA for arachidonic acid metabolites in patients might cause a decrease in airway inflammation and an improvement in clinical asthma. We studied the effect of addition of EPA to the diet of twelve asthmatic patients. Standard clinical evaluations and pulmonary function tests were done on weeks 0, 3, 6, 10, 12 and 14. Patients ingested either low-dose EPA (0.1 g/day) or high-dose EPA (4.0 g/day) from weeks 6-14 (total of 8 weeks). There was no difference in clinical status or pulmonary function between groups at the start of the study. There was no change in clinical status or pulmonary function between or within groups at the end of 8 weeks of EPA ingestion.

Immunological mediators of wound healing and fibrosis.

T-lymphocytes, monocytes, and macrophages, which are the central constituents of immunological and chronic inflammatory reactions, generate numerous polypeptides and other factors capable of stimulating and modulating the proliferation and functions of fibroblasts. These principles differ widely in structure, target cell preference, and functional specificity. The involvement of immunological mediators of fibroblast activities in normal wound healing has not been defined, but a role in some chronic fibrosing disorders, including rheumatoid arthritis, has been suggested by the findings of functionally relevant concentrations in affected tissues. The elucidation of both the pathways of production of fibroblast-activating factors (FAFs) and the determinants of fibroblast responses will permit new approaches to the diagnosis and treatment of deficiencies in wound healing and of abnormal fibrosis.

Structural diversity of the fibroblast-activating factors generated by human blood monocytes and U937 cells.

The incubation of purified human blood monocytes with phytohemagglutinin (PHA) and of cultured U937 human monocyte-like cells with phorbol myristate acetate (PMA) evoked the generation of fibroblast-activating activity, as assessed by stimulation of the uptake of [3H]thymidine by human dermal fibroblasts. Filtration of the supernates from monocytes and U937 cells on Sephadex G-75 resolved fibroblast-activating factors of m.w. 25,000 to 40,000, designated FAF-M and FAF-U937, respectively, from smaller factors of an apparent m.w. of approximately 10,000. FAF-M and FAF-U937 were acidic by isoelectric focusing with respective pI values of 4.0 to 5.2 and 5.4 to 5.6. The smaller factors from both sources filtered on Sephadex G-25 in phosphate-buffered saline with an apparent m.w. of 10,000. However, filtration of the same factors on Sephadex G-25 in 0.1 M acetic acid revealed one predominant fibroblast-activating activity for each cell source of an apparent m.w. of 500 to 1000. The 500 to 1000 dalton factors were inactivated by treatment with trypsin and subtilisin, suggesting that the activity was attributable to fibroblast-activating peptides, termed FAP-M and FAP-U937. FAP-M and FAP-U937 each appeared to be composed of a predominant hydrophilic activity by reverse-phase high-performance liquid chromatography. Human blood monocytes and U937 monocytes both produce structurally diverse fibroblast-activating proteins and peptides, which may contribute to the immunologic regulation of wound healing and fibrosis.

Indomethacin enhances the proliferation of mitogen-stimulated T lymphocytes of homosexual males with persistent generalized lymphadenopathy.

The possibility that cyclooxygenase products of arachidonic acid might contribute to the defective T lymphocyte function of homosexual men with the reactive lymph node syndrome was examined in vitro. T lymphocyte proliferation, assessed by the uptake of [3H]thymidine after the addition of phytohemagglutin, was 72,870 +/- 66,816 counts per minute (mean +/- SD) for eight patients and 119,589 +/- 64,913 counts per minute for 30 controls (P less than 0.05, Student's t test). Treatment with the cyclooxygenase inhibitor indomethacin increased the phytohemagglutin-induced proliferation of the T lymphocytes from five of eight patients, but none of 12 healthy homosexual and heterosexual control subjects. The production of prostaglandin E2 by T lymphocytes from six patients was 16.1 +/- 10.5 pg/5 X 10(6) cells/hr, as contrasted with that of 4.9 +/- 1.3 and 4.3 +/- 2.1 pg/5 X 10(6) cells for four healthy homosexual and six healthy heterosexual control subjects, respectively (P less than 0.01, Student's t test). The production of prostaglandin E2 by the patients' monocytes was normal. Abnormalities of the cyclooxygenase pathway of T lymphocytes of patients with the reactive lymph node syndrome may reflect an immunoregulatory defect, which predisposes to infections and may evolve into the more severe abnormalities of the acquired immune deficiency syndrome.

Generation of a unique fibroblast-activating factor by human monocytes.

Purified human monocytes incubated with phytohemagglutinin (PHA), bacterial lipopolysaccharide (LPS), or serum-opsonized zymosan particles (OZ) generate human dermal fibroblast-activating activity, as assessed by increased fibroblast incorporation of [3H]-thymidine. A maximum concentration of fibroblast-activating activity was attained within 4 hr with OZ, whereas similar maximum levels required 12 hr with LPS and PHA. Sonicates of unstimulated monocytes had only minimal activity and the protein synthesis inhibitor cycloheximide suppressed significantly the appearance of fibroblast-activating activity, suggesting that the factors are generated prior to release. Filtration of supernates from OZ-stimulated monocytes on Sephadex G-75 yielded polydisperse fibroblast-activating activities, of which the major factors exhibited a mol. wt. of approximately 60,000 and 10,000. The supernates from PHA-stimulated monocytes had one predominant factor, termed fibroblast-activating factor of monocytes (FAF-M), with an apparent mol. wt. of 38,000 and a minor activity with a mol. wt. of 10,000. FAF-M was composed of two principles with isoelectric points of 5.1-5.2 and 4.0-4.2 and was free of interleukin-1, as determined by the absence of thymocyte-activating activity. FAF-M and other fibroblast-activating factors may contribute to wound healing and fibrosis in lesions characterized by mononuclear phagocyte infiltrates.

Histamine in human tears.

Tear samples from 13 normal volunteers and nine patients with vernal conjunctivitis were assayed for histamine. Tears in both groups contained histamine. The normal subjects had values ranging from 2.2 to 36 ng/ml with a mean of 10.3 ng/ml. The vernal patients had histamine values ranging from 0 to 125 ng/ml with a mean of 3,.2 ng/ml. The presence of histamine in tears indicates a role for this important mediator in both the physiologic and immunologic processes of the external eye.