D E Chenoweth

C5a and formyl peptide receptor regulation on human monocytes

Regulation of C5a and formyl‐methionine‐leucine‐phenylalanine‐lysine (fMLPL) receptors on human monocytes has been studied using fluorescein‐conjugated derivatives and flow cytometry. Monocytes have receptors for each of these ligands, as evidenced by their ability to bind specifically biologically active fluorescein derivatives of these ligands. Quenching experiments showed that bound fluoresceinated C5a and fMLPL are rapidly internalized at 37°C. Once internalized, monocytes are able to reexpress these receptors, returning to control levels within approximately 90 min. This contrasts with rate differences seen in polymorphonuclear neutrophils (PMNs), where fMLPL receptors return more rapidly (~ 30 min) than do C5a receptors (~ 100 min). Monensin inhibited the reexpression of C5a but not fMLPL receptors, suggesting that a receptor recycling process is necessary to replenish C5a receptors on the monocyte surface. Similar although less efficient inhibition of C5a receptor reexpression was observed with NH4Cl treatment. Reexpression of both G5a and fMLPL receptors was independent of extracellular Ca2+. Treatment with various agents known to stimulate monocytes and PMNs increased the expression of fMLPL receptors in both cell types but either had no effect on or reduced the level of C5a receptor expression. This would indicate that monocytes, like PMNs, have intracellular pools of preformed fMLPL receptors, available for reexpression. These studies show that, like PMNs, monocytes modulate C5a and fMLPL receptors through different mechanisms. Furthermore, monocytes are capable of reexpressing these receptors following exposure to ligand, a theoretical requirement for chemotaxis.

Relationship of chemotactic receptors for formyl peptide and C5a to CR1, CR3, and Fc receptors on human neutrophils.

The co‐expression of C5a and formyl‐methionine‐leucine‐phenylalanine‐lysine (FMLPL) receptors with CR1, CR3, and Fc receptors on human neutrophils (PMN) was studied. Fluorescein‐conjugated C5a (FL‐C5a) and FMLPL (FL‐FMLPL) were used to identify C5a and formyl peptide receptors. CR1, CR3, and Fc receptors were identified with monoclonal antibodies and a Texas red‐labeled goat anti‐mouse immunoglobulin second step reagent The co‐expression of chemotactic receptors with CR1, CR3, or Fc receptors was evaluated using two‐color flow cytometry. A direct correlation between the degree of expression of receptors for FL‐FMLPL and the expression of CR3, CR1, and Fc receptors on individual PMN was observed. In contrast, no correlation between the degree of C5a receptor expression and CR1, CR3, or Fc receptor expression was found. Similar results were obtained with PMN after up regulation of CR1, CR3, Fc, and FMLPL receptors by incubation at 37°C for 10 min with or without phorbol myristate acetate. These data suggest that the expression of FMLPL, CR1, CR3, and Fc receptors are regulated in a similar manner, whereas C5a receptor expression is regulated independently. Furthermore, these data indicate that within a given population of PMN, a parallel exists between the degree of CR1, CR3, FMLPL, and Fc receptor expression on individual cells.

Wheat germ agglutinin inhibits the C5a receptor interaction: implications for receptor microheterogeneity and ligand binding site.

Wheat germ agglutinin (WGA) has been shown to inhibit the interaction of C5a with the C5a receptor on both polymorphonuclear neutrophils (PMNs) and the histiocytic cell line U937. The level of inhibition with isolated receptor preparations is 100%, and on intact cells 10 to 20% of the receptor population appear to retain their ability to bind C5a in the presence of WGA. In contrast, this lectin completely inhibits the C5a‐mediated degranulation of PMN primary and secondary granules, suggesting that the population of C5a receptors responsible for mediating degranulation is also recognized by WGA. More than 50% of the receptors appear to be blocked before an effect on degranulation occurs. This inhibition by WGA does not appear to be due to down‐regulation of C5a receptors from the cell surface, excessive aggregation of receptor sites, or interaction of WGA with the carbohydrate portion of the C5a molecule. The inhibition is reversed by N‐acetylglucosamine but not by sialic acid. This effect appears to be specific for WGA because various other lectins do not inhibit the C5a receptor interaction. That the inhibition by WGA is due to direct binding of the lectin to N‐acetylglucosamine residues on the C5a receptor is strongly supported by the ability of the cross‐linked C5a‐receptor complex to bind to and be specifically eluted from a WGA‐Affigel affinity matrix. These observations are consistent with hypothesis that the population of C5a receptors on leukocytes exhibits microheterogeneity with respect to structure (carbohydrate content) and/or function. J. Leukoc. Biol. 52: 3–10; 1992.

Preliminary Report on Complement Activating Potential of Polycarbonate Membrane

Clinical as well as laboratory studies have been employed to assess the complement activating potential of polycarbonate membrane hemodialyzers. Blood samples from a group of patients undergoing sequential maintenance hemodialysis with cuprophane, polyacrylonitrile and polycarbonate devices were evaluated to define plasma levels of C3a antigen and leukocyte counts during the initial phases of hemodialysis. While polyacrylonitrile dialyzers did not activate complement to a significant extent, we did observe transient elevations in the plasma concentration of C3a and corresponding diminutions in the granulocyte counts of patients dialyzed with both cuprophane and polycarbonate dialyzers. However, polycarbonate devices appeared to activate complement to a lesser degree than cellulosic dialyzers. Laboratory evaluation of these three different types of dialyzers also provided evidence that polycarbonate membranes did not appear to activate human complement as readily as cuprophane. These observations suggest that polycarbonate membranes display complement-related biocompatibility properties that are intermediate between those of cuprophane and polyacrylonitrile.

Immune Response to Reuse: Anaphylatoxins and IgE

Two responses of the immune system have relevance for those interested in artificial kidney performance: first, those associated with complement activation and the release of anaphylatoxins and, second, those associated with immune complex formation and the type-I hypersensitivity reaction. Kaplow and Goffinet in 1972 [1] noted that profound leukopenia occurs within 30 min after initiating cuprophan ® hemodialysis. This phenomenon is now known to be the result of sequestration of polymorphonuclear leukocytes in the pulmonary microvasculative rather than on the dialysis membrane. It was noted early on that not all membranes cause this phenomenon [2, 3]. Increased polymorph adhesiveness is produced when certain types of dialyzer membranes activate the complement system with release of the anaphylatoxin C5a which binds to specific polymorph receptors. The type-I hypersensitivity reaction was brought into focus by the practice of membrane reuse and is considered as the most likely mechanism underlying one form of first-use syndrome.