Granules of the Human Neutrophilic Polymorphonuclear Leukocyte

The phagocytic response of innate immune cells such as macrophages is defined by the activation of complex signaling networks that are stimulated by microbial contact. Many individual proteins have been demonstrated to participate in phagocytosis, and the application of high-throughput tools has indicated that many more remain to be described. In this review, we examine this complexity and describe how during recognition, multiple receptors are simultaneously engaged to mediate internalization, activate microbial killing, and induce the production of inflammatory cytokines and chemokines. Many signaling molecules perform multiple functions during phagocytosis, and these molecules are likely to be key regulators of the process. Indeed, pathogenic microorganisms target many of these molecules in their attempts to evade destruction.

Phagocytosis of Microbes: Complexity in Action

A purified recombinant human granulocyte-macrophage colony stimulating factor (rH GM-CSF) was a powerful stimulator of mature human eosinophils and neutrophils. The purified rH GM-CSF enhanced the cytotoxic activity of neutrophils and eosinophils against antibody-coated targets, stimulated phagocytosis of serum-opsonized yeast by both cell types in a dose-dependent manner, and stimulated neutrophil-mediated iodination in the presence of zymosan. In addition, rH GM-CSF enhanced N-formylmethionylleucylphenylalanine(FMLP)-stimulated degranulation of Cytochalasin B pretreated neutrophils and FMLP-stimulated superoxide production. In contrast, rH GM-CSF did not promote adherence of granulocytes to endothelial cells or plastic surfaces. rH GM-CSF selectively enhanced the surface expression of granulocyte functional antigens 1 and 2, and the Mo1 antigen. rH GM-CSF induced morphological changes and enhanced the survival of both neutrophils and eosinophils by 6 and 9 h, respectively. These experiments show that granulocyte-macrophage colony stimulating factor can selectively stimulate mature granulocyte function.

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Recombinant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival.

Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins.

Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock

The complement system consists of both plasma and membrane proteins. The former influence the inflammatory response, immune modulation, and host defense. The latter are complement receptors, which mediate the cellular effects of complement activation, and regulatory proteins, which protect host cells from complement-mediated injury. Complement activation occurs via either the classical or the alternative pathway, which converge at the level of C3 and share a sequence of terminal components. Four aspects of the complement cascade are critical to its function and regulation: (i) activation of the classical pathway, (ii) activation of the alternative pathway, (iii) C3 convertase formation and C3 deposition, and (iv) membrane attack complex assembly and insertion. In general, mechanisms evolved by pathogenic microbes to resist the effects of complement are targeted to these four steps. Because individual complement proteins subserve unique functional activities and are activated in a sequential manner, complement deficiency states are associated with predictable defects in complement-dependent functions. These deficiency states can be grouped by which of the above four mechanisms they disrupt. They are distinguished by unique epidemiologic, clinical, and microbiologic features and are most prevalent in patients with certain rheumatologic and infectious diseases. Ethnic background and the incidence of infection are important cofactors determining this prevalence. Although complement undoubtedly plays a role in host defense against many microbial pathogens, it appears most important in protection against encapsulated bacteria, especially Neisseria meningitidis but also Streptococcus pneumoniae, Haemophilus influenzae, and, to a lesser extent, Neisseria gonorrhoeae. The availability of effective polysaccharide vaccines and antibiotics provides an immunologic and chemotherapeutic rationale for preventing and treating infection in patients with these deficiencies.

Infectious diseases associated with complement deficiencies.

We used monoclonal antibodies and flow cytometry to study the expression of the receptors for the complement fragments C3bi (CR3) and C3b (CR1) on human polymorphonuclear neutrophil leukocytes (PMN). Expression of both receptors was minimal on cells stained in anticoagulated whole blood incubated at 0 degree or 37 degrees C. PMN isolated with Percoll density gradients and held at 0 degree C also had only minimal expression of both receptors. With the isolated cells, however, a spontaneous increase in expression of both receptors occurred upon warming to 37 degrees C. This did not represent complete expression of either receptor since additional increments in surface expression could be induced upon stimulation with N-formyl-methionyl-leucyl-phenylalanine or Raji cell supernatant. The increases in complement receptor (CR) expression appeared to be specific since there were no changes in expression of the Fc gamma receptor or beta-2-microglobulin under any of these conditions. The increased CR expression seems to involve translocation from an intracellular pool since it is complete within minutes and is not blocked by puromycin or cycloheximide. These results demonstrate that both CR3 and CR1 expression increase rapidly upon activation of PMN and that isolated cells can be used to study this phenomenon, which may be a critical part of neutrophil function in vivo.

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Human neutrophils increase expression of C3bi as well as C3b receptors upon activation.

We have previously shown that complement component 3 (C3) deposited onto encapsulated Streptococcus pneumoniae by anti-capsular antibody (Ab) is a more efficient opsonin in vitro and in vivo than C3 deposited by anti-cell wall Ab (Brown et al., J. Clin. Invest. 69:85-98, 1982). In the present study, we explored the cellular location of C3b molecules that differ in opsonic efficiency by using avidin-ferritin to localize biotinylated Ab and C3 molecules on S. pneumoniae for electron microscopy. Anti-cell wall Ab and C3b molecules deposited by this Ab on unencapsulated S. pneumoniae were localized to S. pneumoniae cell walls. Anti-capsular Ab and C3b deposited by this Ab were seen in clusters on encapsulated S. pneumoniae at a distance from the cell wall. However, no avidin-ferritin staining of encapsulated S. pneumoniae was seen on incubation with biotinyl-anti-cell wall Ab, biotinylated C3 fixed by anti-cell wall Ab, or nonimmune serum containing biotinyl-C3. In each case, uptake of the biotinylated component was proven by radioactivity measurements, since biotinylated Ab and C3 were also radiolabeled with 125I. When avidin-ferritin did not bind to biotinylated components. Ouchterlony analysis indicated that C3 was bound to cell wall components on the encapsulated organisms. Thus, we conclude that, for encapsulated S. pneumoniae, opsonically efficient C3b molecules, deposited by anti-capsular Ab, are located on the S. pneumoniae capsule, whereas the opsonically inefficient C3b molecules deposited by anti-cell wall Ab or nonimmune serum are located on the cell wall. A major reason for the increased virulence of encapsulated compared to unencapsulated S. pneumoniae is that, in the absence of anti-capsular Ab, the S. pneumoniae capsule interferes with the recognition of cell wall-bound C3b molecules by phagocytic cell receptors.

Localization of complement component 3 on Streptococcus pneumoniae: anti-capsular antibody causes complement deposition on the pneumococcal capsule.

It has recently been shown that human neutrophils rapidly increase surface expression of membrane receptors for C3b (CR1) and C3bi (CR3) in response to chemoattractants and other stimuli. In the present studies, we used monoclonal antibodies and flow cytometry to assess the role of Ca2+ in this process. Stimulation with ionophore A23187 in the presence of 1.2 mM Ca2+ increased CR1 330% and CR3 650% compared with unstimulated cells at 37 degrees C. Because this indicated that increasing the cytosolic free Ca2+ caused increased receptor expression, we examined the role of Ca2+ in the response to other stimuli as well. Adding 1.2 mM Ca2+ or 5 mM EDTA to the media in which the polymorphonuclear leukocytes were suspended had no effect on the CR1 response to f-MLP or LTB4, whereas Ca2+ slightly enhanced and EDTA markedly inhibited the CR3 response to these stimuli. The effects of Mg2+-EGTA and EDTA were identical. TMB-8 (200 microM), which inhibits the release of Ca2+ from intracellular stores, completely blocked the increased expression of both receptors induced by fMLP or LTB4. Increased expression of both receptors was also prevented by the calmodulin antagonists chlorpromazine (50 microM) and trifluoperazine (10 microM), but not by chlorpromazine sulfoxide. Phorbol myristate acetate (0.1 ng/ml) increased CR1 230% and CR3 265%. Again Ca2+ and EDTA did not alter the CR1 response, whereas Ca2+ increased CR3 to 287% and EDTA reduced CR3 to 187% of control. TMB-8 (250 microM) completely blocked both CR1 and CR3 responses to this stimulus as well. Thus, release of intracellular Ca2+ is necessary and sufficient for increased CR1 expression in response to diverse stimuli, but maximal increases in CR3 expression require an additional influx of extracellular Ca2+. These results indicate that the mechanisms by which the surface expression of the two different complement receptors increase are different in their requirements for extracellular Ca2+. In comparison with the work of others, we suggest that the processes of increased complement receptor expression and secretion of granular enzymes may also differ.

Calcium requirements for increased complement receptor expression during neutrophil activation.

The binding of serum C3 to the O-antigen capsule (OAg Cap), lipopolysaccharide (LPS), and outer membrane proteins (OMP) of Escherichia coli 0111B4 was examined. Bacteria were intrinsically labeled with [3H] or [14C]galactose (*gal) in the OAg Cap and LPS moieties or with [14C]leucine (*leu) to label proteins. Organisms were then incubated in serum containing differentially labeled C3, the above fractions were separated, and the proportion of each binding to a column containing anti-C3 was measured. The OAg Cap fraction bound 72 to 82% of the C3, which bound to E. coli 0111B4 during incubation in absorbed 10% pooled normal human serum (10% PNHS) or absorbed 40% C8-deficient serum (C8D). This distribution did not change when the organism was presensitized with immune IgG before serum incubation. A total of 2.93% +/- 0.48 of OAg Cap and 0.52% +/- 0.16 of LPS *gal bound specifically to Sepharose-containing antibodies to C3 (A:C3-Seph) after incubation in 10% PNHS; these values increased to 10.1% +/- 4.5 and 1.8% +/- 0.3, respectively, when C3 deposition was increased fourfold by incubation in 40% C8D. When encapsulated E. coli 0111B4 was incubated in 10% PNHS containing biotinylated C3, specific attachment of OAg Cap *gal to avidin-Sepharose was demonstrated in 1% sodium dodecyl sulfate (SDS), and complete release of bound *gal but not C3 occurred with 1 M NH2OH. When a mutant of E. coli 0111B4 lacking OAg Cap was incubated in 40% C8D, the outer membrane (OM) bound 85% of C3. Five percent of OM *gal from the unencapsulated organism bound to A:C3-Seph in 0.05% SDS, indicating that the fraction of LPS molecules with bound C3 increased threefold in the absence of OAg Cap. OAg Cap does not contain protein, and no net specific binding of *leu from OAg Cap fractions to A:C3 was detectable; 2.4 to 3.6% of OM *leu bound to A:C3-Seph. Immunoprecipitation of 82.9% of OAg Cap *gal with antisera that were directed to E. coli 0111B4 was associated with co-precipitation of 69.5% of C3 in the capsular fraction. Therefore, the majority of C3 bound to E. coli 0111B4 was covalently attached to OAg Cap and LPS. As corroboration of experiments with whole bacteria, purified OAg Cap and purified LPS consumed C3 when incubated in serum in the fluid phase. These results are the first to evaluate the acceptor site for C3 deposition on a Gram-negative organism incubated in serum, and show that LPS, OAg Cap, and OMP are all major acceptor sites for C3 in nonimmune serum.

A quantitative analysis of C3 binding to O-antigen capsule, lipopolysaccharide, and outer membrane protein of E. Coli 0111B4

We have examined whether or not a physical relationship exists between antipneumococcal antibodies (Ab) and C3b when Ab activate the classical complement pathway on the surface of pneumococci (Pn). After sensitization with 125I-labeled Ab, Pn were sequentially incubated with purified C1, C4, C2, and biotinylated C3. Ab molecules were then eluted from Pn, and C3b-associated molecules were purified on avidin-Sepharose. Both 125I-labeled immunoglobulin G (IgG) and [125I]IgM bound to C3b; the association was stable to incubation in 1% sodium dodecyl sulfate at 37 degrees C. The association was only partially reversed by incubation in 1 M hydroxylamine-0.5% sodium dodecyl sulfate (pH 10.5), implying that Ab and C3b were linked by amide as well as ester bonds. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dithiothreitol and NH2OH eluates from the avidin-Sepharose showed that C3b bound to both heavy and light chains of the Ab. Moreover, the ability to bind to erythrocyte C3b receptors could be transferred to unsensitized Pn by eluates from Pn on which Ab had activated the classical pathway. These covalent complexes of Ab and C3b may be especially important in the opsonization of Pn by Ab and complement.

https://iai.asm.org/content/iai/42/2/594.full.pdf

M Berger

Papers

Human neutrophils increase expression of C3bi as well as C3b receptors upon activation.

Localization of complement component 3 on Streptococcus pneumoniae: anti-capsular antibody causes complement deposition on the pneumococcal capsule.

Calcium requirements for increased complement receptor expression during neutrophil activation.

A quantitative analysis of C3 binding to O-antigen capsule, lipopolysaccharide, and outer membrane protein of E. Coli 0111B4

Classical complement pathway activation by antipneumococcal antibodies leads to covalent binding of C3b to antibody molecules.