BOTH non-specific immunity and specific humoral immunity mediated by antibodies to bacterial cell envelope components are important host defence mechanisms against invasion by gram-negative bacilli. The presence of depressed levels of polymorphonuclear leucocytes (PMNs) or impaired phagocytic function have been found to correlate with an increased susceptibility to infections (Bodey et al., 1966; Winkelstein and Drachman, 1974). Also the presence of low serum levels of opsonising antibodies directed against bacterial envelope antigens correlate with the occurrence of a high morbidity in septicaemia caused by gram-negative bacilli (Young et al., 1977a; Zinner and McCabe, 1976). There are conflicting reports regarding the specificity of these protective antibodies. The cell envelope of members of the Enterobacteriaceae consists of an outer membrane, containing lipopolysaccharide (LPS) and lipoproteins, and a rigid inner layer containing peptidoglycan. LPS contains the 0 antigen linked to lipid A through a common core structure (Luderitz, Staub and Westphal, 1966). Antibodies against antigenic determinants in the core region have been reported to be protective against the sequelae of bacteremia due to gram-negative bacilli (McCabe, 1972; Young, Stevens and Ingram, 1975). These antibodies seem to act primarily as antitoxins rather than as opsonins (Braude, Douglas and Davis, 1973). Antibodies against 0-antigenic side chains seem to enhance phagocytosis (Medeavaris, Camitta and Heath, 1968; Young, 1974). Several other studies indicate that only capsular polysaccharides (K antigens) have antiphagocytic activity and suggest that strains possessing K antigens have enhanced virulence (Dri et al., 1976; Young et al., 19773). Meanwhile Howard and G l y ~ (1971) demonstrated that K antigens also inhibit the complement-dependent bactericidal activity of serum. Recent workers, however, were unable to find a correlation between the presence of K antigen and the capacity of Escherichia coli to resist phagocytosis and the bactericidal activity of serum (Taylor, 1975; Bjorksten et al., 1976); McCabe et al. (1975) could not demonstrate a relationship between the amount of K antigen produced by different E. coli strains and their virulence in man. The relation between the presence of K antigen and resistance to phagocytosis and the bacteriolytic action of serum has been re-investigated in this study by examination of E. coli strains isolated from faeces and blood.

Neutrophils can be primed by bacterial lipopolysaccharide (LPS) for an enhanced oxidative burst, which is a key element in the pathogenesis of Gram-negative sepsis. Some serum proteins (e.g. lipopolysaccharide-binding protein) avidly bind LPS and markedly enhance receptor binding and cellular activation while other serum factors (lipoproteins, bactericidal/permeability-increasing protein) neutralize LPS and prevent neutrophil activation. In this paper we examined the kinetics of this priming reaction in whole blood. To study the balance between neutrophil activation and LPS neutralization a sensitive chemiluminescence assay was used in a whole blood system. LPS was able to prime neutrophils for enhanced oxidative burst in whole blood with an optimum incubation time of 25 min. However, LPS was neutralized very rapidly with a t(1/2) of 10 min. After 20 min a second priming factor was already generated, which was shown to be monocyte-derived tumour necrosis factor (TNF).

LPS-binding proteins in plasma play an important role in modifying LPS toxicity. Significant properties have already been attributed to the LPS-binding protein (LBP). It accelerates LPS toxicity as well as incorporation into high-density lipoproteins, leading to neutralization of LPS in serum. A search for other LPS-binding components in serum, using LPS-coated magnetic beads, revealed a new LPS-binding protein. N-terminal microsequencing identified this protein as serum amyloid P component (SAP). Purified SAP bound to smooth and rough types of LPS via the lipid A part. SAP inhibited the binding of FITC-labeled ReLPS (LPS from Salmonella minnesota strain R595) to human monocytes and the ReLPS-induced priming of the oxidative burst of human neutrophils only in the presence of low concentrations of LBP. In search for the LPS binding site of SAP, we found that pep27-39, a 13-mer peptide consisting of amino acids 27-39 of SAP, competitively inhibited the binding of LPS to SAP. In addition, pep27-39 significantly inhibited ReLPS-induced responses in phagocytes in the presence of serum, as well as in human whole blood. Carboxamidomethylated pep27-39 showed an even more pronounced reduction of the ReLPS-induced priming of phagocytes in human blood. Performing gel filtration of FITC-labeled ReLPS incubated with soluble CD14, we showed that SAP could not prevent binding of LPS to soluble CD14, in contrast to pep27-39. The ability of pep27-39 to antagonize specifically the effects of LPS in the complex environment of human blood suggests that pep27-39 may be a novel therapeutic agent in the treatment of gram-negative sepsis.

Monocytes produced tumour necrosis factor-alpha (TNF-alpha) upon interaction with infective herpes simplex virus (HSV). Therefore, TNF-alpha and its action were examined in the regulation of anti-viral functions of human polymorphonuclear leucocytes (PMN). The uptake of fluorescein-labelled HSV by human PMN was monitored using flow cytometric analysis. As shown in earlier work, complement-coated herpes virions were bound to PMN but were not internalized. However, after priming with recombinant human TNF-alpha, complement-coated virions were taken up by human PMN. This complement receptor-mediated internalization is a new observation, both because it affects PMN and it is induced by a recombinant cytokine. Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), another priming factor of PMN, was unable to induce this phenomenon. By contrast, other parameters of PMN anti-viral defence were enhanced to the same extent by both TNF-alpha and GM-CSF. PMN primed by either TNF-alpha or GM-CSF showed both an enhanced respiratory burst and an increased membrane potential depolarization when triggered by immune complexes containing HSV, antibody and complement. Both primers rendered an enhanced uptake of HSV in the presence of specific antibody and in the presence of both antibody and complement, but they caused no effect on the rate or quantity of processing of phagocytosed HSV particles by PMN. We propose that TNF-alpha and GM-CSF act as effective modulators of PMH to enhance virus removal, especially in inflammatory sites. We tentatively conclude that TNF-alpha together with PMN and complement represent a novel non-specific defence mechanism against HSV.

The degradation of herpes simplex virus particles after uptake by phagocytes was studied, but, since lysis of the phagocyte also resulted in damage to the viral envelope, measurement of viral infectivity as a criterion of viral degradation after phagocytosis was not possible. Therefore we focused on later events in viral destruction, namely the degradation of macromolecules. We have demonstrated that polymorphonuclear leukocytes (PMN) and monocytes (MN) can rapidly degrade the membrane proteins of the phagocytosed herpes-virus virions. PMN and MN from a patient with chronic granulomatous disease showed a similar rate of degradation compared to PMN and MN from healthy donors, which excludes an important role for toxic oxygen species in viral protein degradation. Experiments using toxic oxygen species-generating systems supported this observation. In contrast to PMN, MN are also effective in the digestion of viral DNA. We conclude that PMN and MN are able to neutralize large amounts of phagocytosed HSV, so their role in antiviral defence has again been demonstrated.

Polymorphonuclear leukocytes (PMN) can mediate cytotoxic reactions against virus infected targets cells. We observed very efficient binding of PMN to HSV-infected fibroblasts when loaded with HSV-specific antibodies. Using electron microscopy, infected fibroblasts were found to be totally surrounded by PMN and the phagocytosis of virions and fragments of infected cells was demonstrated. To quantify and study this phenomenon, and to compare PMN with monocytes, we developed radiometric and fluorometric phagocytosis assays. Leukocytes were mixed with [3H]glucosamine- or FITC-labeled virus and incubated at 37 degrees C. PMN associated radioactivity or fluorescence per cell as measured by flow cytometry was determined. PMN phagocytosis was dependent on the presence of specific anti-HSV antibodies and could be enhanced by addition of complement. Monocytes were also able to phagocytize virions; however, the rate of uptake was less than that for PMN. Under optimal conditions the total amount of herpes simplex particles that could be associated with one PMN or monocyte was about 10,000. PMN and monocytes are capable of phagocytosis of HSV. This may be an important factor in preventing the spread of infection in vivo.

The specific binding of human polymorphonuclear leukocytes (PMN) to antibody-coated target cells was characterized by flow cytometry. PMN were labeled with phycoerythrin-E (PE) via a granulocyte-specific monoclonal antibody (leu-M1) and mixed with fluorescein isothiocyanate-labeled K562 tumor cells sensitized with rabbit antiserum. Specific conjugates were formed as analyzed by two-color fluorescence in a flow cytometer. The formation of stable conjugates was dependent on initiation of contact, temperature, time, and antiserum concentration. Studies with inhibitors implicate that microfilaments, but not microtubules, Ca2+, Mg2+, or energy-dependent processes were a prerequisite for binding of PMN to the antibody-coated target cells. No conjugates were formed when uncoated target cells were used or when the experiment was performed in the presence of protein A, indicating that binding was specifically mediated through Fc receptors (FcR). Monoclonal antibodies against the FcRII and FcRIII were used to address the role of these receptors in conjugation. One of the two anti-FcRIII antibodies and an anti-FcRII antibody effectively prevented conjugation. A monoclonal antibody directed against the common beta-chain of the adhesion molecule family and a combination of antibodies against the alpha-chain of LFA-1 and Mo-1 also blocked conjugation when target cells were sensitized under suboptimal conditions. The antibody against the beta-chain also diminished killing of antibody-coated K562, as measured by chromium release when included in the cytotoxicity assay. These results indicate that flow cytometry permits accurate quantitation and characterization of the binding between PMN and antibody-coated target cells, which in principle, can be prevented by monoclonal antibodies against surface receptors. Binding is primarily established by both the FcRII and FcRIII. Adhesion-associated molecules on the PMN surface contribute to optimal binding.

Natural killer (NK) cells are implicated in host defense mechanisms against infectious diseases and malignancies, and exert a rapid spontaneous cytolysis of various tumour cells and virus-infected cells without prior sensitization or activation (Herberman & Ortaldo, 1981). Human NK cells are a subpopulation of non-adherent, non-phagocytic lymphocytes defined as large granular lymphocytes (Timonen, Ortaldo & Herberman, 1981). NK cells possess a receptor for the Fc region of IgG (Perussia et al., 1984) that enables them to attack antibody-loaded targets, a process called antibody-dependent cell-mediated cytotoxicity (ADCC). The cytotoxic reaction of NK cells can be described as a 'stimulus-secretion' model, divided into three definable steps: binding, triggering for lysis and a killer cell-independent lytic step (Hiserodt, Britvan & Targan, 1982). The killing reaction involves a Ca2+-dependent activation, cytoskeletal rearrangement, activation of the arachidonic acid cascade, release of lysosomal enzymes and a cytotoxic factor(s) (Henkart, 1985) and, possibly, production of reactive oxygen species (Helfand, Werkmeister & Roder, 1982; Roder et al., 1982). The role and involvement of reactive oxygen species is still controversial. To study a possible participation of toxic oxygen species in NK-cell mediated cytotoxicity, we altered target cell anti-oxidant defence mechanisms and measured spontaneous NK-cell mediated cytotoxicity and ADCC reactions against tumour cells. We showed that alteration of target cell anti-oxidant systems had no effect on target cell susceptibility to NK-cell mediated killing. In contrast, the susceptibility of the anti-oxidant-depleted targets to oxygen-dependent polymorphonuclear leucocyte (PMN)-mediated cytotoxicity was increased.

We studied release of leukotriene B4 (LTB4) by human polymorphonuclear leukocytes (PMNs) during phagocytosis of staphylococci in the presence or absence of arachidonic acid. The 12×107 PMNs incubated with 3×109 opsonizedS. aureus and 50μM arachidonic acid released 1.45±0.42 nmol LTB4. No LTB4 was detected after stimulation of PMNs withS. aureus or arachidonic acid by themselves. However, by increasing the concentration of arachidonic acid to 200 or 400μM, 1.22±0.45 and 1.98±0.49 nmol LTB4, respectively, was released by PMNs. The effect of different bacteria-PMN ratios on LTB4 production was also studied. LTB4 varied from 0.3 to 2.0 nmol when bacteria/PMN ratios increased from 5 to 50 (respectively) in the presence of 50 μM arachidonic acid. Thus, phagocytizing PMNs produce LTB4 in the presence of arachidonic acid, and its production is dependent on the number of bacteria phagocytized.

Escherichia coli strains with K capsular polysaccharides are relatively resistant to phagocytosis by polymorphonuclear leukocytes, in contrast to E. coli strains without K antigens. This inhibition of phagocytosis is related to an impaired recognition of the K+ strains by the phagocytes due to ineffective opsonization. All five strains without K antigens were readily phagocytized after opsonization in 5% normal serum, compared with no uptake of the K+ strains. Evidence is presented that the decreased opsonization of the K+ strains in normal serum is caused by a low rate of complement activation of the strains, with subsequent absence of C3b fixation or C3d fixation or both to the cell wall of the bacteria. After removal of the K+ antigens by heating of a K+ E. coli strain, the strain was able to activate complement, to bind C3b or C3d or both, and to become opsonized. Complement was then activated via the classical and alternative pathways, which was comparable to the complement consumption by K- E. coli.