Margrith W. Verghese

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.

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 polyphosphoinositide breakdown. The regulatory model proposed herein may be relevant to other receptors which stimulate polyphosphoinositide metabolism.

A pertussis/choleratoxin-sensitive N protein may mediate chemoattractant receptor signal transduction

Chemoattractant receptors on phagocytic leukocytes utilize a guanine nucleotide regulatory (N) protein to activate phospholipase C and subsequent biological responses. Since pertussis toxin inhibits activation of leukocytes by chemoattractants and ribosylates a ca. 40 kD protein in these cells it had generally been assumed that chemoattractant receptors are coupled to Ni. We now report that human polymorphonuclear leukocytes (PMNs), monocytes, and the myeloid HL-60 and U937 cell lines, but not erythrocytes or bovine brain contain a ca. 40 kD protein which is a substrate for ADP ribosylation by choleratoxin (CT). This N protein, termed Nc for chemotaxis-related N protein, comigrates with the ca. 40 kD PT substrate during one-dimensional gel electrophoresis. In vivo treatment of PMNs with PT or CT reduced high affinity binding of chemoattractants to membrane preparations from the cells, implying that chemoattractant receptors are coupled to an N protein which is a substrate for both PT and CT. We suggest that Nc rather than Ni couples chemoattractant receptors to phospholipase C.

Function and regulation of chemoattractant receptors

Phagocyte migration and activation at sites of inflammation is mediated through chemoattractant receptors that are coupled to G-proteins. Early studies from our laboratory demonstrated G-protein-mediated phospholipase C activation by chemoattractants. Recently, this laboratory developed cellular and animal models to allow biochemical, cell biological and molecular genetic approaches to be used in determining the mechanisms of chemoattractant receptor function, regulation, and cross regulation. These studies provided evidence that chemoattractant receptors activate distinct pathways for chemotaxis and exocytosis and cross-regulate each others function at multiple levels. A major site of regulation is through phosphorylation of receptors by G-protein-coupled receptor kinases and by protein kinase C. In addition, the activation of phospholipase C by chemoattractants is also regulated, at additional sites distal to receptor phosphorylation. These may include modulation of G-protein activation by regulators of G-protein signaling (RGS) and modification of phospholipase C. Phosphorylation of phospholipase Cβ3 by both protein kinase A and protein kinase C has been demonstrated. The function and regulation of chemoattractant receptors are also being examined in mouse models. In these studies, mice deficient in leukotriene B4 receptors have been generated by targeted gene disruption. These mice displayed reduced neutrophil accumulation in certain inflammation models and sex-related differences in platelet-activating-factor induced anaphylaxis.

Signal transduction in cells following binding of chemoattractants to membrane receptors

SummaryBinding of chemoattractants to specific cell surface receptors on human polymorphonuclear leukocytes (PMNs) initiates a variety of biologic responses, including directed migration (chemotaxis), release of Superoxide anions, and lysosomal enzyme secretion. Chemoattractant receptors belong to a large class of receptors which utilize the hydrolysis of polyphosphoinositides to initiate Ca2+ mobilization and cellular activation. Receptor occupancy leads to phospholipase C-mediated hydrolysis of polyphosphoinositol 4,5-bisphosphate (PIP2) yielding inositol 1,4,5-trisphosphate (IP3) and 1,2 sn-diacylglycerol (DAG). These products synergize to initiate cell activation via calcium mobilization (IP3) and protein kinase C activation (DAG). Pertussis toxin, which ADP-ribosylates and inactivates some GTP binding proteins (G proteins), abolishes all chemoattractant-induced responses, including Ca2+ mobilization, IP3 and DAG production, enzyme secretion, superoxide production and chemotaxis. Direct evidence for chemoattractant receptor: G protein coupling was obtained using PMN membrane preparations which contain a Ca2+-sensitive phospholipase C. Hydrolysis of polyphosphoinositides at resting intracellular Ca2+ levels (100 nm) was only observed when the membranes were stimulated with the chemoattractant N-formyl-methyl-leucyl-phenylalanine (fMet-Leu-Phe) in the presence of GTP. Myeloid cells contain two distinct pertussis toxin substrates of similar molecular weight (40 and 41 kD). The 41 kD substrate resembles Gi whereas a 40 kD substrate is physically associated with a partially purified fMet-Leu-Phe receptor preparation and may therefore represent a novel G protein involved in chemoattractant-stimulated responses. Metabolism of 1,4,5-IP3 to inositol proceeds via two distinct pathways in PMNs: (1) degradation to 1,4-IP2 and 4-IP1 or (2) conversion to 1,3,4,5-IP4, 1,3,4-IP3, 3,4-IP2 and 3-HV Initial formation (0-30 s) of 1,4,5-IP3 and DAG occurs at ambient intracellular Ca2+ levels, whereas formation of 1,3,4-IP3 and a second sustained phase of DAG production (30 s–10 min) require elevated cytosolic Ca2+ influx. The later peak of DAG, which is not derived from phosphoinositides, appears to be required for stimulation of respiratory burst activity. Products formed during activation can feed back to attenuate chemoattractant receptor-mediated stimulation of phospholipase C by uncoupling receptor-G protein-phospholipase C interaction.

Lymphocyte suppressor activity in patients with polyglandular failure

The Con-A--activated suppressor function of lymphocytes from polyglandular failure (PGF) patients in human mixed lymphocyte culture was compared to a normal population. As a group, PGF patients were found to have decreased suppressor activity: 67% of normal for autologous suppression, 45% of normal for heterologous. However, lymphocytes from most PGF patients have neither an absolute lack of suppressor activity nor an absolute inability to respond to suppression. The marked variability of assayed suppression, depending on the combination of stimulators and responder cells tested, limit the utility of this assay in defining individuals with abnormal suppressor function. One patients lymphocytes were unique in that although suppression of heterologous cells was normal, suppression of her own cells was defective. Defective suppressor function may be related to the susceptibility of these patients to multiple autoimmune diseases.

Murine sera cytotoxicity toward human b cells and their effect on human mixed lymphocyte reactions.

Seven murine anti-H-2 and three nonimmune mouse sera were tested for cytotoxicity toward B and T lymphocytes from a panel of human donors. One group of sera, including two anti-H-2.33 sera, exhibited cytotoxicity directed exclusively toward human B but not T cells from all donors. Absorption studies on human lymphoblastoid cell lines (LCLs) of B or T cell origin corroborated these findings. Some nonimmune sera showed similar characteristics, indicating that the observed reactions were not attributable to cross-reactivities between mouse H-2K or D specificities and human antigens coded by the HLA-A, B, or C locus. Another set of mouse sera (anti-H-2.28b and anti-H-2.31) was highly cytotoxic to both B and T cells of some donors but not of others, suggesting that activity in these sera may arise from cross-reactions between mouse and human specificities. A third set of anti-H-2 as well as normal mouse sera showed only background cytotoxicity when tested on human cells. The ability of the B cell cytotoxic mouse sera to block the human mixed lymphocyte culture reaction (MLR) was compared to that of appropriate human alloantisera with exclusive B cell activity or a rabbit serum raised against human B cells. None of the mouse sera resulted in a significant reduction in the human MLR, whereas the human alloantisera as well as the rabbit antiserum caused a significant amount of blocking at several dilutions beyond their highest cytolytic titer. In an extensive search for cross-reactivity between specificities of the mouse and human major histocompatibility complex (MHC), Ivaskova et al. (1) found that many congenic H-2 sera react with almost all human cells if the serum dilutions are sufficiently low. Associations between some HLA locus antigens (mainly A2, B7, and B27) and certain of the H-2 sera can reportedly be detected only when end point titrations are carried out. The authors point out that there is considerable variability in the strength of reactions of human cells with the mouse alloantisera and that reproducibility of typings is not as good as with HLA alloantisera. This may indicate that some of the antibodies in the H-2 antisera may actually be reacting with subpopulations of human lymphocytes. The proportions of B and T cells from different donors seem to vary from bleed to bleed. This variability could result in different end titration points in sera containing mostly B cell antibodies. The reactivity of the HLA specificities with well defined typing sera usually varies little. This is to be expected, since HLA antigens are present on all lymphocytes. Therefore, some of the differences in reaction strengths observed when donors carrying similar HLA antigens are tested with heterologous antisera may be attributable to reactions involving only certain lymphocyte subpopulations. A preliminary investigation in this laboratory indicated that human LCLs, with B cell characteristics are much more sensitive to H-2 alloantisera than those classified as T cells. The present study demonstrates that some mouse sera exhibit cytotoxic patterns toward separated human B and T cells that closely resemble those of xenoantibodies produced against human B cell lines. The effect of the mouse sera on the human MLR is also investigated. The blocking activity of the mouse sera is, however, much weaker than that of xenoantibodies or human alloantibodies recognizing DRw specificities.

[25] Biochemical changes in leukocytes in response to chemoattractants

Publisher Summary This chapter discusses various biochemical changes in leukocytes in response to chemoattractants. Activation of phagocytic leukocytes by chemoattractants and most other inflammatory stimuli is associated with rapid increases in intracellular [Ca 2+ ]. Optimal stimulation of the secretory and respiratory burst responses of these cells to chemoattractants requires Ca 2+ in the extracellular medium. Chemoattractants stimulate the release of the Ca 2+ mobilizer inositol 1,4,5-trisphosphate (IP 3 ) and 1,2-diacylglycerol in leukocytes as a consequence of phospholipase C (PLC) activation and hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ). The products resulting from PIP 2 hydrolysis stimulate Ca 2+ /phospholipid- and/or calmodulin-dependent protein kinases, which apparently lead to cellular activation. Measurements of Ca 2+ mobilization, cyclic adenosine monophosphate (cAMP) levels, and protein kinase redistribution in polymorphonuclear leukocytes (PMNs) have provided useful tools for studying the response of these cells to mediators of inflammation. Methods for measuring Ca 2+ redistribution commonly utilize the Ca 2+ -sensitive fluoro probes Quin 2-tetraacetomethoxy ester and chlortetracycline. Information about cAMP measurement in intact cells is provided.