A synthetic lipopolysaccharide-binding peptide based on amino acids 27-39 of serum amyloid P component inhibits lipopolysaccharide-induced responses in human blood.

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.

Modulation of lipopolysaccharide binding to human granulocytes.

Using flow cytometry and fluorescein-labelled lipopolysaccharide (LPS) from Salmonella minnesota R595 (FITC-ReLPS), we studied the role of membrane proteins in the recognition of LPS by human polymorphonuclear granulocytes (PMN) in the absence of serum. Treatment of PMN with trypsin, pronase E or proteinase K reduced both the binding of FITC-ReLPS to PMN at 4 degrees and the response of PMN to LPS at 37 degrees, as measured by luminol-enhanced chemiluminescence. Neuraminidase treatment enhanced both activities. Trypsin treatment of PMN after the binding of FITC-ReLPS effectively reduced fluorescence when cells were kept at 4 degrees, while further incubation of FITC-ReLPS-labelled PMN at 37 degrees rendered fluorescence insensible to trypsin. These results indicate a protein structure of the LPS binding site, association of FITC-ReLPS with the cell membrane at 4 degrees and subsequent internalization at 37 degrees. The binding of FITC-ReLPS was not inhibited by the anti-CD14 monoclonal antibody (mAb) 3C10, which recognizes a functional epitope of CD14. Furthermore, binding of FITC-ReLPS was observed to PMN obtained from a patient with paroxysmal nocturnal haemoglobinuria who lacked membrane-bound CD14. Stimulation of PMN with tumour necrosis factor (TNF) or LPS enhanced the binding of FITC-ReLPS at 4 degrees. This was not observed after activation of PMN devoid of granules (cytoplasts), indicating that the binding of LPS at the cell surface is enhanced by mobilization of LPS-binding proteins from intracellular granules. These studies provide evidence that LPS binding and activation of PMN involves protein structures at the cell surface different from CD14, and that granules constitute a pool of LPS-binding proteins that can be translocated to the cell surface upon stimulation.

Tumour necrosis factor triggers granulocytes to internalize complement-coated virus particles.

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.

Degradation of herpes simplex virions by human polymorphonuclear leukocytes and monocytes.

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.

Quantitation of conjugate formation between human polymorphonuclear leukocytes and antibody-coated target cells by flow cytometry: the role of Fc receptor and LFA-1 antigen.

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.

Complement-mediated phagocytosis of herpes simplex virus by granulocytes. Binding or ingestion.

The role of complement receptors in phagocytosis of herpes simplex virus (HSV) by PMN was examined. Complement components were deposited on the surface of the virus particle in the presence or absence of specific anti-HSV antibodies. Flow cytometry was used to analyze the phagocytosis of fluorescence-labeled viruses and demonstrated that although a virion is able to associate with PMN in the presence of complement alone, the granulocyte is not triggered to mount a metabolic burst. Efficient stimulation of PMN occurs when complexes are formed consisting of virus, specific antibodies, and complement. To address the question whether the viruses were inside or outside the cell, a combined enhancement/quenching method was developed using ammonium chloride as a lysosomotropic agent and trypan blue as a quenching dye. The data indicate that Fc receptor-mediated phagocytosis by PMN results in the ingestion of all cell-associated herpes virions. Interactions of virions through PMN-complement receptors CR1 and CR3 results solely in binding to the PMN but not in internalization. Interactions via both complement and Fc receptors cause synergistic stimulation of the PMN and result in very efficient association of viruses, greater than 80% of which were inside the cell.

Phagocytosis of herpes simplex virus by human granulocytes and monocytes.

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.

Further evidence against a role for toxic oxygen products as lytic agents in NK cell-mediated cytotoxicity.

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.

Human polymorphonuclear leukocytes release leukotriene B4 during phagocytosis of Staphylococcus aureus.

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 X 10(7) PMNs incubated with 3 X 10(9) opsonized S. aureus and 50 microM arachidonic acid released 1.45 +/- 0.42 nmol LTB4. No LTB4 was detected after stimulation of PMNs with S. aureus or arachidonic acid by themselves. However, by increasing the concentration of arachidonic acid to 200 or 400 microM, 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 microM arachidonic acid. Thus, phagocytizing PMNs produce LTB4 in the presence of arachidonic acid, and its production is dependent on the number of bacteria phagocytized.

Interactions between human polymorphonuclear leukocytes and influenza virus.

The effects of influenza virus A (H3N2) on several functions of human polymorphonuclear leukocytes (PMN) were examined. Incubation of PMN with virus induced chemiluminescence, aggregation, and degranulation of the leukocytes. The amount of chemiluminescence generated increased from 1 X 10(6) to 6 X 10(6) cpm when 2.5 X 10(6) to 2 X 10(7) virus particles were added to 2.5 X 10(6) PMN. Maximal aggregation occurred within 2 min and the response depended on the amount of virus added to the PMN. Release of acid phosphatase by virus-treated PMN was 62 +/- 12% within 1 h compared with 7 +/- 7% by control PMN (P less than 0.005). Incubation of PMN with influenza virus resulted in a diminished phagocytic activity of the phagocytes. PMN from a patient with chronic granulomatous disease were similarly affected. It was thus concluded that the observed defect in phagocytic activity was not due to the reactive oxygen species generated by the PMN during incubation with virus.

Differences in the effect of arachidonic acid on polymorphonuclear and mononuclear leukocyte function.

Incubation of human polymorphonuclear leukocytes with arachidonic acid resulted in a stimulation of the oxidative metabolism of the cells. Upon stimulation with 80 microM arachidonic acid, neutrophils (5 X 10(6) cells/ml) produced superoxide (53 +/- 8 nmol/5 X 10(6) cells per 15 min), generated chemiluminescence (1211 100 +/- 157 000 cpm) and consumed oxygen (20 +/- 1 nmol/10(6) cells per 5 min). The stimulation of the cell metabolism could be reduced 40-60% by prior incubation of the cells with 10 microM indomethacin. Incubating polymorphonuclear leukocytes with arachidonic acid also resulted in a diminished chemotaxis towards an attractant, a decreased uptake of opsonized staphylococci and aggregation of the cells. This may be due to inhibitory products of arachidonic acid metabolism and toxic oxygen species produced during stimulated oxidative metabolism. The effects of arachidonic acid are specific for neutrophils, as mononuclear phagocytes only produced 17 +/- 8 nmol superoxide/5 X 10(6) cells per 15 min and generated 27 000 +/- 15 000 cpm chemiluminescence when stimulated with 80 microM arachidonic acid. When monocytes and neutrophils were stimulated with particles such as opsonized staphylococci, the amount of superoxide produced, oxygen consumed and chemiluminescence generated were similar. The phagocytic activity of the monocytes was also not affected by prior incubation with arachidonic acid. We conclude that in contrast to monocytes, neutrophil metabolism can be stimulated with arachidonic acid and this stimulation resulted in a decreased phagocytic activity of these cells.

Aggregation of human polymorphonuclear leucocytes during phagocytosis of bacteria.

The process of aggregation of human polymorphonuclear leucocytes (PMN) during the uptake of bacteria was studied. Radiolabelled S. aureus were opsonized in different sera, washed, resuspended in buffer and added to the PMN. Uptake of the bacteria and aggregation of the PMN were measured simultaneously. Maximal aggregation occurred within 6 min, when 5 X 10(6) PMN had phagocytosed 2.5 X 10(8) S. aureus. Also the effects of serum concentrations and different sera for opsonization of the bacteria on PMN aggregation were studied. Despite normal uptake, aggregation of PMN was low when bacteria were opsonized in complement-deficient sera. Furthermore when PMN were treated with pronase to inactivate complement receptors on the cell surface of the PMN, and bacteria preopsonized in immune serum were added, no change in uptake occurred, although the degree of aggregation halved compared to control PMN. So, interaction between the bacteria and the complement receptor of the PMN cell membrane is needed for triggering the process of aggregation. By using dansylcadaverin and diphenylamine to modulate lysosomal enzyme release, azide or PMN from a chronic granulomatous disease patient to study the effect of the formation of oxygen species, and theophylline, DB-cAMP or 8 Br-cAMP to increase cAMP levels, it was concluded that aggregation of PMN during phagocytosis was not dependent on oxygen metabolism, degranulation or cAMP levels of PMN.

Escherichia coli lipopolysaccharides diminish and enhance cell function of human polymorphonuclear leukocytes.

The effects of the lipopolysaccharide (LPS) of Escherichia coli J5 and 0111B4 on the function of human polymorphonuclear leukocytes (PMN) were tested. E. coli J5 is a UDP-galactose-4-epimerase-deficient mutant of E. coli 0111B4, and its LPS, therefore, contains mainly lipid A, as it lacks the polysaccharide side chains. PMN which had been incubated with J5 LPS showed decreased phagocytic, chemotactic, and metabolic activities as compared with control PMN. In contrast, incubation of PMN with 0111B4 LPS had no effect or even an enhancing effect on PMN function. When lipid A and the polysaccharide fraction were isolated from 0111B4 LPS, it was shown that lipid A had the same deleterious effect on PMN function as did J5 LPS and that the LPS fraction had no effect. When PMN were incubated with J5 LPS or lipid A, it could be shown that these structures were able to induce PMN to generate superoxide and chemiluminescence. 0111B4 LPS and the polysaccharide component were able to generate a metabolic burst by the PMN to a lesser extent. The induced defects in PMN function by J5 LPS could be prevented when polymyxin B or an oxygen-radical scavenger was present. We hypothesize that the lipid A portion of LPS is toxic for PMN due to the induction of toxic oxygen species by the PMN. These toxic oxygen species destroy the phagocytic, chemotactic, and metabolic activities of the PMN.

Escherichia coli antibodies in opsonisation and protection against infection.

The opsonic and protective capacities of rabbit antisera against Escherichia coli O, K and core-glycolipid cell-wall antigens were compared with specific antibody titres as measured by agglutination and enzyme-linked immunosorbent assay. Anti-O antisera were opsonic and protective against two noncapsulate strains. Only anti-K antisera were opsonic and protective against a K-antigen-containing strain. In a mouse model anti-core-glycolipid antiserum was not protective against challenge even by a strain bearing only core glycolipid.

Interactions of phagocytic and bacterial cells in patients with bacteremia caused by gram-negative rods.

The phagocytic and bactericidal functions of polymorphonuclear leukocytes and monocytes and the opsonic activity of serum from patients with gram-negative bacteremia were compared with those of cells and serum from healthy donors and control patients. Leukocytes from five of 20 patients showed diminished phagocytic capacity. Leukocytes from three of 12 patients had decreased chemotactic activity. Eleven of 37 blood culture isolates were inefficiently phagocytized after opsonization in homologous patient serum. However, in no instance was the opsonic capacity of patient serum significantly lower than that of control serum. In the serum of some patients, an increase in heat-stable opsonins was found during the course of infection. Resistance to opsonization of strains of Escherichia coli correlated with the presence of K capsular polysaccharide. It was concluded that both impaired leukocyte function and ineffective opsonization play a role in the pathogenesis of gram-negative bacteremia. Heat-stable opsonins (presumably specific antibodies) appear to be necessary for effective phagocytosis of bacilli that cause gram-negative bacteremia.

Role of Escherichia coli K capsular antigens during complement activation, C3 fixation, and opsonization.

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.

The role of Staphylococcus aureus cell-wall peptidoglycan, teichoic acid and protein A in the processes of complement activation and opsonization.

The role of cell-wall peptidoglycan, teichoic acid and protein A in the processes of Staphylococcus aureus complement activation and opsonization was investigated. CH50 consumption studies reveal that, although all cell-surface fractions were capable of activating the classical C pathway, only peptidoglycan consumed C via the alternative pathway. Using a quantitative immunofluorescence assay, peptidoglycan was shown to bind C3 molecules via the classical as well as via the alternative C pathway and in the absence of IgG and IgA class antibodies. C activation via the classical and the alternative pathway could be distinguished by kinetic analysis. By comparing the rates of staphylococcal C consumption, C3 fixation and opsonization it was found that the CH50 consumption assay is a relatively insensitive method and may yield results that do not necessarily reflect the process of bacterial opsonization.

Staphylococcus aureus opsonization mediated via the classical and alternative complement pathways. A kinetic study using MgEGTA chelated serum and human sera deficient in IgG and complement factors C1s and C2.

Staphylococcus aureus opsonization was studied kinetically by: (1) determination of the uptake of [3H]-thymidine labelled bacteria by human PMN's; (2) fluorescent anti-C3 and anti-IgG staining of opsonized bacteria; and (3) measuring bacterial complement consumption. Maximum opsonization in normal serum occurred within 5 min of incubation. About 80% of staphylococci were then taken up by PMN's, and IgG and C3b could be detected on the bacterial surface. In the absence of a functional classical complement pathway, as in sera deficient in C1s and C2 and in MgEGTA chelated serum, maximal opsonization was only achieved after 30--60 min incubation. Opsonization in IgG deficient serum occurred at a rate similar to that found in C2 deficient or MgEGTA chelated serum. Opsonization was greatly enhanced when sera were reconstituted. It was concluded that in IgG deficient serum Staphylococcus aureus opsonization is mediated via the alternative complement pathway. Dilution of normal serum primarily affected the classical complement pathway, resulting in a decreased rate of opsonization. In normal serum IgG did not appear to be a rate-limiting factor. S. Aureus opsonization was best studied by the phagocytosis assay and the fluorescent-antibody technique. Measuring haemolytic complement consumption was found to be an insensitive indicator of bacterial complement activation and opsonization.