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Biomedical subjects

R Snyderman

Publications and source records attributed to R Snyderman.

At least 109 records · Page 6Linked to original sources
PubMed1986

Role of guanine nucleotide regulatory protein in polyphosphoinositide degradation and activation of phagocytic leukocytes by chemoattractants.

Leukocyte activation by chemoattractants provides an important model to study the biochemical mechanisms of stimulus-response coupling in these cells. Well-defined chemotactic factors induce readily quantifiable responses in phagocytic leukocytes. These include directed migration and the production and release of toxic substances including oxygen radicals and lysosomal enzymes. The development of radiolabeled synthetic oligopeptides with potent chemotactic activity allowed the demonstration of chemoattractant receptors on polymorphonuclear leukocytes (PMNs) as well as macrophages. In membrane preparations from these cells, these receptors exist in high- and low-affinity states which are regulated by guanosine di- and triphosphates. This suggested that chemoattractant receptors interact with guanine nucleotide regulatory proteins (N or G proteins). Although chemoattractants elicit a rapid but transient increase in intracellular cAMP levels, they neither stimulate nor inhibit membrane-bound adenylate cyclase, suggesting a novel role for N proteins in certain receptor-transduction mechanisms. Stimulation of phagocytes by chemoattractants is also associated with a rapid increase in cytosolic Ca2+ concentrations ([ Ca2+]i) which appears to result from the production of inositol 1,4,5-triphosphate (IP3) as a consequence of the diesteric cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2). Treatment of phagocytes with pertussis toxin (PT), which ADP-ribosylates and thereby inactivates certain N proteins, abolishes the cells' responsiveness to chemoattractants. More direct evidence for a role of a PT-sensitive N protein in leukocyte activation was provided by the demonstration that chemoattractants stimulate the hydrolysis of PIP2 in PMN membranes only in the presence of GTP. This receptor-mediated hydrolysis of PIP2 is not observed in plasma membranes prepared from PT-treated PMNs. Therefore, these studies suggest that occupancy of chemoattractant receptors activates a PT-sensitive N protein. The activated N protein shifts the Ca2+ requirement for phospholipase C activity from supraphysiological levels to ambient cytosolic Ca2+ concentrations. Cleavage of PIP2 results in the formation of the second messenger molecules, IP3 and 1,2-diacylglycerol, which can initiate cellular activation. These messengers also seem to activate responses which feed back to attenuate receptor stimulation of phospholipase.

Adenylyl Cyclases↗
PubMed1986

Model for leukocyte regulation by chemoattractant receptors: roles of a guanine nucleotide regulatory protein and polyphosphoinositide metabolism.

Binding of chemoattractants to their receptors on phagocytes activates a guanine nucleotide regulatory (N) protein through the substitution of GTP for GDP on N. The activated N protein in turn stimulates a membrane-associated phospholipase C by lowering the Ca2+ concentration required to activate this enzyme from supraphysiologic levels to ambient intracellular concentrations. The phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate into the Ca2+ mobilizer inositol 1,4,5-trisphosphate and the protein kinase C activator 1,2-diacylglycerol. In addition to promoting cellular activation, the products of this hydrolysis initiate processes which feed back to inhibit poly-phosphoinositide breakdown. The regulatory model proposed herein may be relevant to other receptors which stimulate polyphosphoinositide metabolism.

Adenylyl Cyclases↗
PubMed1986

Effects of tumor growth on host defenses.

Monocytes and macrophages play an important role in host defense against neoplasia. Studies from our and other laboratories have demonstrated that patients with a variety of cancers have a defect in monocyte chemotactic responses. Tumor-bearing mice are also inhibited in their ability to accumulate macrophages to inflammatory foci. We have shown that extracts prepared from murine tumors, as well as the plasma and urine of tumor-bearing mice, contain anti-inflammatory proteins which are antigenically and physicochemically related to the immunosuppressive retroviral envelope protein p15E. Similarly, proteins capable of inhibiting monocyte chemotactic responses are present in human cancerous effusions and can be specifically absorbed by monoclonal antibodies to p15E. Furthermore, we have demonstrated that human malignant and mitogen-transformed cells contain p15E-related antigens. These findings led us to propose a two stage model of tumorigenesis: the first stage involves neoplastic transformation of a cell while the second stage involves activation of a gene coding for a p15E-like protein which allows the transformed cell to escape immune surveillance and go on to become a tumor. Support for this model has come from recent studies which have identified within the human genome an endogenous retrovirus sequence whose envelope gene is partially homologous to a highly conserved region of p15E. Using a synthetic peptide, termed CKS-17, we have shown that this region may be responsible for many of the biological activities of p15E and is capable of suppressing lymphocyte and natural killer cell immune functions as well as those of monocytes and macrophages. Thus tumors may be capable of evading host defense mechanisms by activation of a normal gene related to the immunosuppressive retroviral protein p15E.

Animals↗
PubMed1986

Mechanisms of inflammation and leukocyte chemotaxis in the rheumatic diseases.

Understanding the mechanisms of inflammatory cell accumulation, as well as how such cells mediate tissue destruction, provides better insights into the pathogenesis and therapeutics of the rheumatic diseases. This article discusses the role of the inflammatory process in normal immune function, the mechanisms of inflammatory cell accumulation, and how the local accumulation of inflammatory cells can lead to the clinical signs and symptoms associated with rheumatic disorders.

Antibodies↗
PubMed1986

Chemoattractant-mediated stimulation of the respiratory burst in human polymorphonuclear leukocytes may require appearance of protein kinase activity in the cells' particulate fraction.

Low doses of aliphatic alcohols produce divergent effects on the function of chemoattractant receptors on human polymorphonuclear leukocytes (PMNs) since they enhance chemotaxis but inhibit stimulation of superoxide production by chemoattractants. As such, alcohols can provide useful pharmacologic tools to probe the mechanisms of stimulus-response coupling in leukocytes. A role for protein kinase C has been implicated in the activation of the respiratory burst in PMNs. Although the vast majority of this enzyme activity is located in the cytosolic fraction of unactivated PMNs, protein kinase C activity appears in the particulate fraction of the cells when they are stimulated to produce superoxide by either chemoattractants or by phorbol myristate acetate (PMA). Doses of the alcohols that selectively inhibited stimulation of superoxide production by chemoattractants also inhibited the appearance of protein kinase C activity as well as an undefined protein kinase activity in the particulate fraction of the cells. In contrast, the alcohols did not affect either the ability of PMA to stimulate the production of superoxide in PMNs nor the appearance of protein kinase activity in the cells' particulate fraction. PMA is known to bind and activate protein kinase C directly, thus bypassing receptor-mediated events. These data suggest that alcohols inhibit the stimulation of the respiratory burst by chemoattractants in PMNs by blocking the ability of receptor occupancy to induce the appearance of protein kinase activity in particulate fractions. These results moreover suggest that the appearance of protein kinase activity in the particulate fraction may be required for activation of the respiratory burst in PMNs.

Alcohols↗
PubMed1985

Inhibition of lymphocyte proliferation by a synthetic peptide homologous to retroviral envelope proteins.

The retroviral transmembrane envelope protein p15E is immunosuppressive in that it inhibits immune responses of lymphocytes, monocytes, and macrophages. A region of p15E has been conserved among murine and feline retroviruses; a homologous region is also found in the transmembrane envelope proteins of the human retroviruses HTLV-I and HTLV-II and in a putative envelope protein encoded by an endogenous C-type human retroviral DNA. A peptide (CKS-17) was synthesized to correspond to this region of homology and was examined for its effects on lymphocyte proliferation. CKS-17 inhibited the proliferation of an interleukin-2-dependent murine cytotoxic T-cell line as well as alloantigen-stimulated proliferation of murine and human lymphocytes. Four other peptides, representing different regions of virus proteins, were inactive. These results suggest that the immunosuppressive portion of retroviral transmembrane envelope proteins may reside, at least in part, in a-conserved sequence represented by the CKS-17 peptide.

Amino Acid Sequence↗
PubMed1985

Chemoattractant-elicited alterations of cAMP levels in human polymorphonuclear leukocytes require a Ca2+-dependent mechanism which is independent of transmembrane activation of adenylate cyclase.

The affinity of the chemoattractant receptor for N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) on human polymorphonuclear leukocytes (PMNs) is regulated by guanine nucleotides, and chemoattractants stimulate increased intracellular cAMP levels in PMNs. Our data, however, indicate that this receptor does not activate membrane-bound adenylate cyclase via direct nucleotide regulatory protein (N) coupling but instead raises cAMP levels indirectly via a mechanism which appears to require Ca2+ mobilization. This conclusion is based on the following data: 1) prostaglandin E1 (PGE1) activated and alpha 2-adrenergic treatment inhibited adenylate cyclase activation in PMN plasma membranes; fMet-Leu-Phe, however, neither activated nor inhibited adenylate cyclase in these membranes; 2) depletion of extracellular Ca2+ had no effect on isoproterenol and PGE1 elicited cAMP responses in intact PMNs while peak fMet-Leu-Phe and A23187-induced responses were reduced by approximately 50 and 80%, respectively; 3) 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, a purported Ca2+ antagonist, caused almost complete inhibition of fMet-Leu-Phe and ionophore-induced cAMP responses in intact cells but had no effect on PGE1 and isoproterenol; 4) alpha 2-adrenergic agonists inhibited PGE1 but not chemoattractant- or A23187-elicited cAMP responses in intact PMNs; and 5) pretreatment of cells with a phosphodiesterase inhibitor (isobutylmethylxanthine) greatly potentiated the PGE1 and isoproterenol cAMP responses but nearly abolished the peak fMet-Leu-Phe response. Thus, chemoattractants appear to utilize a novel mechanism to raise cAMP levels which appear to require Ca2+ mobilization and could be mediated in part through a transient inhibition of phosphodiesterases. We suggest that stimulation of PMN functions by chemoattractants may utilize an N-coupled process to generate a Ca2+ signal which could in turn raise intracellular cAMP levels indirectly and thereby provide negative regulation.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Chemoattractant receptor-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate in human polymorphonuclear leukocyte membranes. Requirement for a guanine nucleotide regulatory protein.

Incubation of plasma membranes from human polymorphonuclear leukocytes (PMNs) with [gamma-32P]ATP in the presence of MgCl2 resulted in the formation of 32P-labeled phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). Membranes from PMN specific and azurophil granules synthesized only PIP, suggesting that PIP2 metabolism is confined to the plasma membrane in PMNs. Further incubations of the labeled plasma membranes for 60 s in the presence of 1 mM CaCl2 resulted in the hydrolysis of approximately 40 and 50% of the labeled PIP and PIP2, respectively. In the presence of 2 microM added CaCl2, PIP and PIP2 levels were unchanged by incubation with either the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) at 0.1 microM or by 10 microM GTP; however, addition of fMet-Leu-Phe plus GTP together resulted in a 11 and 28% decrease in PIP and PIP2, respectively. These treatments had no effect on PA levels. No additional radiolabeled organic-soluble products were detected after treatment with fMet-Leu-Phe plus GTP. Incubation of intact PMNs, with the Bordetella pertussis toxin (islet-activating protein) eliminated the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in the isolated plasma membranes, but did not inhibit PIP2 degradation in the presence of 1 mM CaCl2. These results provide the first direct evidence that the fMet-Leu-Phe receptor in PMN membranes is coupled to polyphosphoinositide hydrolysis through an islet-activating protein-sensitive guanine nucleotide regulatory protein.

Bacterial Toxins↗
PubMed1985

Potential role for a guanine nucleotide regulatory protein in chemoattractant receptor mediated polyphosphoinositide metabolism, Ca++ mobilization and cellular responses by leukocytes.

Islet activating protein from Bordetella pertussis toxin which ribosylates certain guanine nucleotide regulatory proteins causes a marked reduction of chemoattractant-elicited responses such as chemotaxis, O2 production and cAMP elevations in human polymorphonuclear leukocytes. The toxin appears to exert its effects by preventing the rapid breakdown of phosphatidylinositol 4,5-bisphosphate induced by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, thereby inhibiting the increase in intracellular [Ca++] which normally follows chemoattractant stimulation. Responses of leukocytes exposed to Concanavalin A, the Ca++ ionophore A23187, or phorbol myristate acetate were not affected by the toxin. Thus the chemoattractant receptor appears to be coupled to a phosphoinositide specific phospholipase C through a guanine nucleotide regulatory protein. We propose that this complex of receptor-guanine nucleotide regulatory protein-phospholipase C may be applicable to the class of receptors which mobilize intracellular Ca++ by stimulating polyphosphoinositide breakdown.

Bacterial Toxins↗
PubMed1985

Pneumonitis complicating low-dose methotrexate therapy in rheumatoid arthritis.

Three of 95 patients with rheumatoid arthritis who were being treated with low-dose (5 to 15 mg/wk) methotrexate sodium developed the clinical, radiographic, and pathologic features of methotrexate-associated pulmonary injury. Marked hypoxemia emphasized the severity of illness in our patients; lowest oxygen pressure values for each patient were 35 mm Hg, 42 mm Hg, and 45 mm Hg. The management of our patients with a pulmonary toxic reaction to methotrexate included discontinuing the drug treatment, antibiotic therapy until an infectious cause was excluded, and high-dose methylprednisolone. Two patients recovered and one died. Contrary to an earlier report that suggested that pneumonitis occurred only with methotrexate sodium doses exceeding 15 mg/wk, our three cases demonstrate that a severe pulmonary toxic reaction may also complicate low-dose weekly methotrexate therapy of rheumatoid arthritis.

Arthritis, Rheumatoid↗
PubMed1985

Activation of the respiratory burst enzyme from human neutrophils in a cell-free system. Evidence for a soluble cofactor.

Activation of the respiratory burst in phagocytic cells, an important host defense process, is not yet well understood. We now report the development of a cell-free system for activation of NADPH oxidase, the respiratory burst enzyme, in human neutrophils. Activation was achieved by the addition of arachidonic acid to a postnuclear supernatant (500 g) from disrupted unstimulated cells (no arachidonate, 0.2; with arachidonate, 3.4 nmol superoxide anion/min per mg) and was dependent on both the concentration of arachidonate and on the amount of cellular material present. Activity stimulated by arachidonate appeared to be NADPH oxidase based on a Michaelis constant for NADPH of 32 microM and a pH optimum of 7.0-7.5. Separation of the 500-g supernatant by high speed centrifugation revealed a requirement for both soluble and particulate cofactors. Activation of NADPH oxidase by arachidonate did not occur in the high speed pellet fraction from unstimulated cells but could be restored by the addition of the high speed supernatant. In addition, priming of intact neutrophils with low concentrations of the chemoattractant N-formyl-methionyl-leucyl-phenylalanine or the tumor promoter phorbol myristate acetate replaced the soluble factor requirement for NADPH oxidase activation by arachidonate in the high speed pellet. This cell-free system can now be used to provide further insight into the biochemical basis of priming and the terminal mechanisms involved in the activation of NADPH oxidase.

Arachidonic Acid↗
PubMed1985

Redistribution of protein kinase C activity in human monocytes: correlation with activation of the respiratory burst.

Protein kinase C (PKC) was found to be present in purified human monocytes and lymphocytes isolated by countercurrent centrifugal elutriation. In unstimulated monocytes and lymphocytes, approximately 90% of the PKC activity was cytosolic when the cells were disrupted in the presence of EGTA. The role of this kinase in the stimulation of the respiratory burst in monocytes was investigated. Phorbol esters capable of triggering the release of O2- caused a loss of PKC activity from the cytosol and the appearance of the kinase activity in the particulate cell fraction. Kinase activity was partially extractable from the particulate fraction by 0.1% Triton X-100, whereupon it demonstrated calcium and lipid dependence. The EC50 for the phorbols in initiating the respiratory burst correlated well with their EC50 for stimulating the appearance of PKC activity in the particulate fraction (R = 0.998). Redistribution of PKC activity in monocytes by phorbol myristate acetate (PMA) was rapid and appeared to precede the release of O2-. PMA also shifted PKC activity from the cytosol to the extractable particulate fraction of lymphocytes. We conclude that redistribution of PKC activity by active phorbols or other cell stimulants could provide substrate specificity for phosphorylation reactions. By shifting PKC activity to the monocyte particulate fraction, active phorbols may initiate the phosphorylation of a substrate required for stimulation of the respiratory burst.

Cell Separation↗
PubMed1985

Phorbol myristate acetate mediates redistribution of protein kinase C in human neutrophils: potential role in the activation of the respiratory burst enzyme.

Protein kinase C may be important in leukocyte function, because it is activated by phorbol myristate acetate (PMA), a potent stimulus of the respiratory burst in neutrophils. The localization of protein kinase C was compared in unstimulated and PMA-stimulated human neutrophils. Protein kinase C was primarily cytosolic in unstimulated cells but became associated with the particulate fraction after treatment of cells with PMA. The particulate-associated kinase activity did not require added calcium and lipids, but when extracted by Triton X-100 (greater than or equal to 0.2%), calcium and phospholipid dependence could be demonstrated. The EC50 of PMA for stimulating kinase redistribution and activation of NADPH oxidase, the respiratory burst enzyme, were similar (30 to 40 nM). Redistribution of protein kinase C occurred rapidly (no lag) and preceded NADPH oxidase activation (30 sec lag). These results suggest that redistribution of protein kinase C is linked to activation of the respiratory burst in human neutrophils.

Alkaline Phosphatase↗
PubMed1985

Inhaled asbestos activates a complement-dependent chemoattractant for macrophages.

Pulmonary macrophages migrate to the sites where inhaled chrysotile asbestos fibers initially are deposited (i.e., surfaces of alveolar duct bifurcations). These macrophages have been shown to form a major component of an early asbestos-induced interstitial lesion in rats. In this report, we describe a potential mechanism by which macrophages are attached to these sites of fiber deposition. Chrysotile asbestos fibers used in vitro activate complement proteins in peripheral blood serum and in lavaged cell-free lung proteins. After brief inhalation of chrysotile asbestos, fluids lavaged from the lungs of exposed rats contain substantial chemotactic activity for macrophages compared to fluids from sham-exposed animals (p less than 0.01). We hypothesize that this chemotactic activity is derived from complement activated by inhaled asbestos on alveolar surfaces. This contention is supported by the following observations. Production of chemotactic activity by asbestos in vitro in serum or in lavaged lung fluids was blocked by complement inhibitors. Fractionation, by molecular sieve chromatography, of serum proteins and concentrated proteins lavaged from the lungs of asbestos-exposed rats showed that chemotactic activity was detected in the 14,000- to 18,000-dalton range. This fractionation profile is similar to C5a, the chemotactic product of complement activation. Rats treated with cobra venom factor to deplete circulating complement as well as complement-deficient mice demonstrated significantly depressed macrophage accumulation at sites of asbestos deposition. Pulmonary macrophages are the cells that form the initial inflammatory response to asbestos inhalation. Our findings support the hypothesis that asbestos fibers, and possibly other inhaled particulates, activate complement-derived chemotactic activity on alveolar surfaces. Consequently, macrophages are attracted to the alveolar duct bifurcations where inhaled asbestos fibers are deposited, and this is where the initial lesion of asbestosis is manifested.

Animals↗
PubMed1985

Successful tumour immunotherapy: possible role of antibodies to anti-inflammatory factors produced by neoplasms.

Phenol-saline tumour extracts, active in the immunotherapy of bovine ocular squamous cell carcinoma (BOSCC), were used to immunize mice. The immunized mice became resistant to the depression of delayed type hypersensitivity (DTH) reactions by products of BOSCC cells or cultured mouse or rat tumour cells. They also showed partial resistance to the growth of an unrelated tumour. Monoclonal antibodies to a type C retrovirus protein, p15E, also induced resistance to the depression of DTH by products of BOSCC and mouse tumours. It is suggested that successful immunotherapy of BOSCC is associated with the induction of resistance to anti-inflammatory or immunosuppressive tumour cell products, allowing the operation of host defences, and that these products share antigenic determinants with a retrovirus protein.

Animals↗