Induced Neutrophil Chemotaxis Research Articles

The CXC-Chemokine Neutrophil-Activating Peptide-2 Induces Two Distinct Optima of Neutrophil Chemotaxis by Differential Interaction With Interleukin-8 Receptors CXCR-1 and CXCR-2

AbstractThe CXC-chemokines interleukin-8 (IL-8), neutrophil-activating peptide-2 (NAP-2), and melanoma growth-stimulatory activity (MGSA) are chemoattractants with high selectivity for neutrophils. Although IL-8 has been shown to act as an extremely potent mediator, reports on NAP-2 and MGSA are still contradictory. Here we show for the first time that NAP-2 and MGSA induce two distinct optima of neutrophil chemotaxis. A first optimum is elicited within a concentration range as low as it is characteristic for IL-8. However, a second optimum appears at more than 200-fold higher stimulus concentrations, at which IL-8 is inactive. Investigating the involvement of the two chemokine receptors CXCR-1 and CXCR-2 in NAP-2–mediated chemotaxis, we observe that the cells become desensitized to the first optimum of the chemokine after selective downregulation of CXCR-2, while both optima disappear upon simultaneous downregulation of both receptors. Blocking monoclonal antibodies (MoAbs) specific for CXCR-2 or CXCR-1 either suppress the first optimum of NAP-2–induced chemotaxis or drastically reduce the second one, respectively. These results provide evidence that both receptors are involved in NAP-2–induced neutrophil chemotaxis, with CXCR-2 rendering the cells responsive to low dosages of the chemokine, and with CXCR-1 extending their responsiveness to NAP-2 dosages higher by several orders of magnitude.

Granulocyte chemotactic protein-2 and related CXC chemokines: from gene regulation to receptor usage.

Chemokines contribute to the inflammatory response by selective attraction of various leukocytic cell types. Human GCP-2 was originally identified by amino acid sequence analysis as a CXC chemokine co-produced with IL-8 by osteosarcoma cells. Furthermore, the complete coding domain of human GCP-2 was disclosed by means of RT-PCR. Similarly, mouse GCP-2 was isolated from fibroblastoid and epithelial cells and completely identified by sequence analysis. Human and mouse GCP-2 share 61% identical amino acids. Both chemokines occur as multiple NH2-terminally truncated forms. The shorter forms of mouse, but not those of human, GCP-2 showed a higher neutrophil chemotactic potency and gelatinase B releasing capacity. Mouse GCP-2 was a more potent neutrophil activator than human GCP-2, natural mouse KC, and MIP-2. Human GCP-2 was not chemotactic for monocytes, lymphocytes, or eosinophils. Quantitative studies of mRNA expression in diploid fibroblasts revealed GCP-2 induction by IL-1beta. Human GCP-2 induced [Ca2+]i increase in neutrophils, which was reciprocally desensitized by IL-8, GROalpha, and ENA-78. Human GCP-2 induced [Ca2+]i increases and chemotactic responses in both CXCR1- and CXCR2-transfected cells. Finally, GCP-2 provoked neutrophil accumulation and plasma extravasation in rabbit skin. In humans, GCP-2 complements the activity of IL-8 as neutrophil chemoattractant and activator but it constitutes a major neutrophil chemokine in the mouse. GCP-2 induces neutrophil chemotaxis and activation but it might also contribute to detrimental tissue damage in sepsis, acute respiratory distress syndrome, acute hypersensitivity reactions, and autoimmune diseases. It might also influence the invasive capacity of GCP-2-secreting tumor cells.

The involvement of macrophage-derived tumour necrosis factor and lipoxygenase products on the neutrophil recruitment induced by Clostridium difficile toxin B.

Clostridium difficile (Cd) toxins appear to mediate the inflammatory response in pseudomembranous colitis and/or colitis associated with the use of antibiotics. In contrast to Cd Toxin A (TxA), Cd Toxin B (TxB) has been reported not to promote fluid secretion or morphological damage in rabbits and hamsters and also does not induce neutrophil chemotaxis in vitro. However, TxB is about 1000 times more potent than TxA in stimulating the release of tumour necrosis factor-alpha (TNF-alpha) by cultured monocytes. In the present study, we investigated the ability of TxB to promote neutrophil migration into peritoneal cavities and subcutaneous air-pouches of rats. We also examined the role of resident peritoneal cells in this process as well as the inflammatory mediators involved. TxB caused a significant and dose-dependent neutrophil influx with a maximal response at 0.1 microgram/cavity after 4 hr. Depleting the peritoneal resident cell population by washing the peritoneal cavity or increasing this population by pretreating the animals with thioglycollate blocked and amplified the TxB-induced neutrophil migration, respectively. Pretreating the animals with MK886 (a lipoxygenase inhibitor), NDGA (a dual cyclo- and lipoxygenase inhibitor) or the glucocorticoid, dexamethasone, but not with indomethacin (a cyclo-oxygenase inhibitor), or BN52021 (a platelet-activating factor antagonist), inhibited the neutrophil migration evoked by TxB. Pretreatment with dexamethasone or the administration of anti-TNF-alpha serum into the air-pouches also significantly reduced the TxB-induced neutrophil migration. Supernatants from TxB-stimulated macrophages induced neutrophil migration when injected into the rat peritoneal cavity. This effect was attenuated by the addition of either MK886 or dexamethasone to the macrophage monolayer and by preincubating the supernatants with anti-TNF-alpha serum. TxB also stimulated the release of TNF-alpha by macrophages. Overall, these results suggest that TxB induces an intense neutrophil migration which is mediated by macrophage-derived TNF-alpha and lipoxygenase products.

Chemoattractant properties of PR-39, a neutrophil antibacterial peptide.

The proline-arginine (PR) -rich antibacterial peptide, PR-39, kills bacteria by a non-pore-forming mechanism. Because this neutrophil peptide possesses several distinct functional properties and because other antimicrobial peptides are chemoattractants, we sought to determine whether PR-39 was a chemoattractant for porcine leukocytes. The peptide was synthesized by the solid-phase method using t-Boc chemistry and purified by reversed-phase high-performance liquid chromatography. Leukocyte migration was assessed with the use of a 48-well microchemotaxis chamber. PR-39 induced the directed migration of neutrophils. The peak chemotaxis response occurred at 0.5-2 microM, which was slightly lower than the minimal inhibitory and bactericidal concentrations for PR-39. However, the peptide was not a chemoattractant for mononuclear cells. Truncation of PR-39 suggested that the neutrophil chemoattractant domain may be contained within the first 26 amino acid residues. Intracellular Ca2+ fluxes in response to PR-39 were monitored by flow cytometry and showed a transient increase that peaked at approximately 40 s and approached basal values by 4 min. However, in a Ca2+-free environment, the PR-39-induced Ca2+ increase was abrogated. Furthermore, PR-39 did not induce neutrophil chemotaxis in the absence of extracellular Ca2+, and pertussis toxin inhibited both neutrophil chemotaxis and Ca2+ mobilization. Taken together, these data suggest that PR-39 is a Ca2+-dependent chemoattractant of neutrophils. The finding of a neutrophil antibacterial peptide that is also a neutrophil chemoattractant is intriguing and may indicate an important role for PR-39 in inflammation.

Neutrophil chemotactic activity in bronchoalveolar lavage fluid of patients with AIDS-associated Pneumocystis carinii pneumonia.

Pneumocystis carinii pneumonia (PCP) is accompanied by an acute inflammatory infiltration of the lung parenchyma. The cellular infiltrate is characterized by inflammatory cells including neutrophils, lymphocytes and macrophages. Furthermore, neutrophilia in bronchoalveolar lavage (BAL) fluid has been shown to confer a poor prognosis in PCP. We therefore investigated the potential of BAL fluid from 17 patients with PCP to induce neutrophil chemotaxis. BAL fluid from patients induced considerable neutrophil chemotactic activity compared to normal controls. Elevated levels of IL-8 were detected in patient samples as compared to controls. A specific anti-IL-8 antibody significantly reduced chemotactic activity of patient samples by more than 50%. In conclusion, IL-8 appears to be a significant participant of neutrophil chemotaxis in AIDS-associated PCP, and may participate in the recruitment of neutrophils to the lung during PCP.

IL-8 induces neutrophil chemotaxis predominantly via type I IL-8 receptors.

IL-8 is a potent proinflammatory cytokine that has a key role in the recruitment and activation of neutrophils during inflammation. IL-8 reacts with neutrophils via two distinct types of IL-8-R. Receptor-specific Abs were raised against peptides derived from the first extracellular domain of each IL-8-R. Anti-IL-8-R1 and anti-IL-8-R2 selectively block 125I-IL-8 binding to rIL-8-R type 1 or 2, respectively. The anti-peptide Abs were used to assess the role of each receptor in the chemotactic response of neutrophils to GRO alpha and to IL-8. Anti-IL-8-R2 blocks GRO alpha-induced chemotaxis of neutrophils. Chemotaxis to GRO alpha is not inhibited by anti-IL-8-R1. Thus GRO alpha stimulates chemotaxis exclusively through IL-8-R2 and independently of IL-8-R1. Surprisingly, anti-IL-8-R1 inhibits the majority (78 +/- 3%) of IL-8-induced neutrophil chemotaxis. Only a minor proportion of IL-8-induced chemotaxis (29 +/- 5%) is inhibited by anti-IL-8-R2. These findings indicate that chemotaxis to IL-8 is mediated predominantly by type 1 IL-8-Rs and suggest that IL-8-R1 is an appropriate target for therapeutic strategies to limit neutrophil influx in diseases where neutrophils contribute to pathophysiology.

Chemical Synthesis, Purification and Folding of the Human Monocyte Chemotactic Proteins MCP-2 and MCP-3 into Biologically Active Chemokines

Monocyte chemotactic proteins 2 and 3 (MCP-2 and MCP-3) are chemokines structurally and functionally related to MCP-1. In contrast to MCP-1, they are produced in low amounts by stimulated leukocytes or tumour cells. As an alternative method to generate sufficient protein for in vitro and in vivo characterization of MCP-2 and MCP-3, we have synthesized both 76-residue chemokines using Fmoc chemistry. After automated solid phase peptide synthesis at a 0.05 to 0.25 mmol scale, and purification to homogeneity by C-8 RP-HPLC, correct disulfide bridges were formed in a mixture of oxidized and reduced glutathione. The synthesis was biochemically controlled by peptide sequencing of intermediate products and proteolytic fragments of the 76-residue chemokines and by mass analysis. Purified synthetic MCP-2 and MCP-3 coeluted and comigrated with their natural counterparts on analytical reverse phase columns and SDS-PAGE, respectively. Purified and folded MCP-2 and MCP-3 were chemotactic for monocytes at 7.5 ng/ml and 5 ng/ml, respectively. These minimal effective concentrations are comparable to those of the natural chemokines. Synthetic MCPs did not induce neutrophil chemotaxis. Automated Fmoc peptide synthesis is thus a useful method, allowing fast production of chemokines and analogues thereof.

Proinflammatory properties of molluscum bodies.

Molluscum contagiosum, a condition characterized by benign viral tumours, occasionally becomes inflamed and regresses spontaneously, an event probably initiated by a host cell-mediated immune rejection against the lesion, but it inevitably involves the disruption of the epidermal tissue to expose the molluscum bodies to the tissue fluids of the dermis. It has been suggested that the molluscum bodies induce inflammation by a mechanism similar to that involved in ruptured epidermal cysts or in acne. Despite the occasional development of inflammation in molluscum contagiosum, the proinflammatory properties of molluscum bodies have never been studied in vitro. Thus, in the present study we sought to determine whether molluscum bodies exert a proinflammatory effect by inducing neutrophil chemotaxis. When exposed to fresh serum in vitro, water-insoluble components of molluscum bodies activated the alternative complement pathway to produce chemotactic C5a/C5a des Arg. We also found that an aqueous extract of molluscum bodies exerted potent chemotactic activity for neutrophils. Remarkably high amounts of the immunoreactive proinflammatory cytokines IL-8 and GRO alpha were present in the extract even when compared with psoriatic scale extracts. Gel filtration HPLC of the extract demonstrated the presence of neutrophil chemotactic activity over a wide range of molecular mass. These data suggest that disruption of the epidermal wall of molluscum bodies induces acute inflammatory changes by activation of the alternative complement pathway on exposure to the tissue fluids, and that the molluscum bodies themselves release proinflammatory cytokines and other neutrophil chemotactic factors on decomposition.

Lipoxygenase-derived mediators may be involved in in vivo neutrophil migration induced by Bothrops erythromelas and Bothrops alternatus venoms

Bothrops erythromelas (BEV) and B. alternatus (BAV) venoms induced a dose-dependent neutrophil migration when injected into rat peritoneal cavities (20–160 μg/cavity). These venoms (80 μg/rat) also induced neutrophil migration in the air pouch model of inflammation. This migratory response seemed to be related to the phospholipase A 2 (PLA 2) activity of the venoms. BAV had approximately two times more PLA 2 activity than BEV, and the neutrophil migration induced by the former venom was two to three-fold greater than that observed with the latter. Heated (90°C for 5 min) BEV lost about 50% of its PLA 2 activity and this was accompanied by a corresponding loss in the ability to induce neutrophil chemotaxis. Dexamethasone (0.5 mg/kg, s.c.), an indirect inhibitor of PLA 2 activity, also abolished the neutrophil migration induced by both venoms. Since NDGA (100 mg/kg, s.c.) and dexamethasone, but not indomethacin (2 mg/kg, s.c.), strongly reduced the neutrophil migration induced by both bothropic venoms, it is suggested that arachidonate-derived lipoxygenase metabolites such as leukotriene B 4 act as the chemotactic mediators. Macrophages could be the main cellular source of such metabolites since they are the predominant resident cells in the rat air pouch, and the migratory response of BEV and BAV into peritoneal cavities was potentiated in rats pretreated with thioglycollate. The neutrophil migration induced by BEV and BAV was not due to endotoxin contamination since heated BEV showed no effect and polymyxin B-treated BAV still remained active.

Elevated levels of NAP-1/interleukin-8 are present in the airspaces of patients with the adult respiratory distress syndrome and are associated with increased mortality.

The adult respiratory distress syndrome (ARDS) is characterized by increased neutrophils within the airspaces of the lungs. In order to determine if neutrophil activating protein (NAP)-1/interleukin-8 (NAP-1/IL-8) could be an important cause of neutrophil influx and activation in ARDS, we examined fluid, which was either directly aspirated or lavaged with saline from the lungs of patients with ARDS. NAP-1/IL-8 was present in significantly higher concentrations in the fluids of patients with ARDS compared with control subjects. There was a significant correlation between the percentage of neutrophils in the lavage fluids and the NAP-1/IL-8 concentration (r2 = 0.74). Furthermore, the NAP-1/IL-8 concentration of the pulmonary edema fluid was equivalent to the optimal concentration required to induce neutrophil chemotaxis in vitro. Although not all of the chemotactic activity of the edema fluid was removed by an anti-NAP-1/IL-8 affinity column, the data established that NAP-1/IL-8 is an important neutrophil chemotaxin in the airspaces of patients with ARDS. In addition, those patients with very high concentrations of NAP-1/IL-8 in their bronchoalveolar lavage fluids had a higher mortality rate than those patients with lower concentrations of NAP-1/IL-8. The correlation between NAP-1/IL-8 concentration and mortality is not paralleled by total protein concentration and mortality.

The detection of a novel neutrophil-activating peptide (ENA-78) using a sensitive ELISA.

Neutrophil elicitation into tissue is an essential element of the host defense in response to various stimuli, including, tissue injury, infection, or cancer. This event has gained renewed interest with the discovery of a family of small polypeptides (less than 10 kD). The salient features of these cytokines are the presence of four cysteine amino acids (first two separated by one amino acid; C-X-C) and their ability to induce neutrophil chemotaxis and activation. Recently, our laboratories have discovered a new member of this C-X-C chemotactic cytokine supergene family, neutrophil-activating peptide, ENA-78. ENA-78 shares significant amino acid sequence homology with neutrophil activating peptide-2 (NAP-2; 53%), growth regulated oncogene/melanoma growth stimulatory activity (GRO alpha; 52%), and IL-8 (22%). In addition, ENA-78 appears to activate neutrophils through the IL-8 receptor. Since both in vitro and in vivo biological fluids may contain an array of chemotactic cytokines that may be relevant to the activation and chemotaxis of neutrophils, we have developed a highly specific and sensitive sandwich ELISA for the detection of ENA-78.

Degree of platelet activating factor-induced neutrophil migration is dependent upon the molecular species.

Multiple molecular species of platelet activating factor (PAF) are produced as a result of inflammatory processes. PAF-induced neutrophil migration across endothelium is intrinsic to inflammatory responses. We therefore compared the ability of three naturally occurring PAF species (C16:0, C18:0, and C18:1), which only varied at carbon 1, to induce 51Cr-labeled human neutrophil migration across a naked 3-microns pore filter and human umbilical vein endothelial (HUVE) monolayers cultured on these filters. Time-course experiments indicated that all species of PAF tested induced significant neutrophil migration between 15 and 45 min. PAF-induced neutrophil migration through both filters alone and HUVE monolayers occurred at lower doses with C16:0 PAF. The rank order of chemotactic potency for the PAF species was C16:0 greater than C18:0 greater than C18:1 with both filters and endothelium coated filters as barriers. Intrinsic differences in the potency of these PAF molecular species to induce neutrophil chemotaxis were greater when HUVE cells were the barriers vs when filters alone were the barriers. Regardless of the molecular species used, at optimal PAF doses the degree of neutrophil migration through HUVE cells was often greater than that through filters alone. The specific PAF antagonist WEB 2086 inhibited neutrophil migration induced by all three PAF species equally. WEB 2086 pretreatment of the neutrophil or WEB 2086 coincubation with PAF, but not WEB 2086 pretreatment of the HUVE cell monolayers, significantly inhibited (65 to 80%) neutrophil migration. We conclude that the degree of PAF-induced neutrophil migration is dependent upon the molecular species of PAF. Moreover, the barrier through which the neutrophil must migrate appears to be important in influencing the overall chemotactic response to the various PAF species.

Interaction of extracellular vesicles of Bacteroides gingivalis W50 with human polymorphonuclear leucocytes

The effects of B. gingivalis W50 extracellular vesicles (ECV) on neutrophil chemotaxis and viability were assessed and compared with those of whole cells and the extracellular non-dialysable soluble protein (EP) fraction. None of the fractions tested, including soluble fractions derived from cells and ECV by sonication, induced neutrophil chemotaxis. Only ECV and cells inhibited f-MLP-stimulated chemotaxis. ECV and cells were cytotoxic towards neutrophils. The cytotoxic response was time dependent. The soluble EP fraction did not influence cell viability.

Inflammatory events after fibrin microembolization. Alterations in alveolar macrophage and neutrophil function.

We performed bronchoalveolar lavage (BAL) 0.5 to 24 h after thrombin-induced pulmonary microembolization in spontaneously breathing sheep to examine the inflammatory events that occur after pulmonary intravascular coagulation. Neutrophil alveolitis was evident as early as 0.5 h after microembolization and was maximal at 4 h (4.9 +/- 1.5% neutrophils of total BAL cells at baseline versus 26.2 +/- 2.8% at 4 h post-thrombin). Neutrophils obtained both at baseline (isolated from peripheral blood) and at 0.5 to 24 h after thrombin (isolated from BAL) did not demonstrate significant basal production of superoxide anion (O2-) and produced similar amounts of O2- upon challenge with phorbol myristate acetate (PMA) 200 micrograms/ml. The basal O2- production by alveolar macrophages was also not increased. However, alveolar macrophages recovered after fibrin microembolization produced greater amounts of O2- (29.1 +/- 6.3 nm O2-/10(6) cells at 0.5 h) after challenge with PMA compared with alveolar macrophages recovered prior to embolization (10.6 +/- 1.6 nm O2-/10(6) cells baseline), suggesting that thrombin-induced microembolization primes alveolar macrophages and enhances their O2- generation. Neutrophil chemotactic activity was detected in BAL fluid at 0.5 h post-microembolization and reached a peak level at 2 h. Alveolar macrophages were a source of the chemotactic activity since conditioned medium obtained from 2-h post-thrombin macrophages induced neutrophil chemotaxis, whereas baseline cells did not. The addition of the thrombin to macrophages did not result in the generation of chemotactic activity from baseline macrophages, indicating that macrophages were activated during the process of intravascular coagulation rather than by thrombin per se. Post-thrombin BAL fluid also stimulated O2- generation from sheep neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

The ability of uremic serum to induce neutrophil chemotaxis in relation to hemodialysis.

The ability of serum to attract normal neutrophil granulocytes was investigated in 11 uremic patients before and after hemodialysis using a modified assay system for granulocyte migration under agarose. The chemotactic responses towards serum from the uremic patients were significantly decreased (p less than 0.01) prior to hemodialysis compared to control serum from a healthy individual. Hemodialysis acutely normalized the depressed chemotactic responses. It is concluded that hemodialysis improves the ability of uremic serum to attract neutrophils, and it is suggested that this improvement might be due to removal of granulocyte inhibitory factors from the sera.

Endotoxin inhalation induces neutrophil chemotaxis by alveolar macrophages.

Guinea-pigs were exposed to an aerosol of bacterial endotoxin (lipopolysaccharide, LPS). The free lung cell response and alveolar macrophage (AM) chemotaxis were studied. Neutrophils from guinea-pig blood gave larger migration responses than those obtained by intraperitoneal glycogen stimulation or human neutrophils. An increase in the number of neutrophils in the airways was found with a peak at 12-24 hours after exposure. In animals pre-treated with LPS inhalation for 4 months, the reaction was of shorter duration and smaller magnitude. AM showed in vitro chemotactic activity up to 4 hours after exposure; no difference was found in pre-treated animals. The results suggest that the neutrophil invasion in the airways after LPS is dependent on two mechanisms, the initial being AM chemotaxis, which is not modified by pre-exposure to LPS, and another unknown factor, which is modified by pre-exposure to LPS.

Neutrophil chemotactic responses induced by fresh and swollen Rhizopus oryzae spores and Aspergillus fumigatus conidia.

With the induction of germination, Rhizopus oryzae spores and Aspergillus fumigatus conidia activate the complement system and induce neutrophil chemotaxis. In contrast, freshly isolated R. oryzae spores did not induce neutrophil migration into lung tissue of mice after intranasal inoculation. Moreover, in microchemotaxis assays neither fresh R. oryzae spores nor A. fumigatus conidia activated sera to stimulate human neutrophil chemotaxis above control migration until at least 10(7) or 10(8) spores or conidia per ml of sera were used. The increased generation of chemotactic factors by swollen spores and conidia was not due to an increased surface area, as there was decreased neutrophil chemotactic response to Rhizopus or Aspergillus hyphae when compared with swollen spores or conidia.

Development of chemotactic factor inactivation - a fetal lamb model.

Summary: To study the developmental pattern of chemotactic factor in-activator (CFI) activity, although avoiding the effects of labor and delivery, serial blood samples were obtained from eight chronically catheterized lamb fetuses, 13 pregnant, and 13 nonpregnant ewes. The biologic profiles of adult sheep neutrophils in response to sheep C5-derived chemotactic fractions (C5-fr) were determined by the chemotactic and lysosomal enzyme release assays. CFI activity in sheep plasma was characterized and measured by the % inhibition of C5-fr-induced neutrophil lysosomal enzyme release. The chemotactic and enzyme release profiles of sheep neutrophils paralleled those of human neutrophils. Sheep plasma was shown to contain a CFI-like activity similar to that in human plasma. Mean CFI activity was higher in the plasma of pregnant ewes as compare to nonpregnant ewes (7.60 versus 5.15%, P < 0.001). Higher CFI levels (39.5%) were shown in the plasma of chronically catheterized fetuses early in the third trimester of pregnancy (109 days gestational age). These fetal levels decreased progressively to attain normal adult levels at fetal maturity (147 days gestational age) and no significant changes were noted at or after birth. Because identical changes occurred in all chronically catheterized fetuses, a developmental pattern was defined for CFI. Control CFI levels from six acutely catheterized fetuses did not differ from those predicted by the developmental pattern. Thus, the changes in CFI activity were not related to the chronic catheterization or to events surrounding labor and delivery; rather CFI levels correlated best with fetal immaturity. CFI levels correlated inversely with ability of zymosan activated plasma to induce neutrophil chemotaxis (r = —0.96, P < 0.01), suggesting that CFI plays a major role in the regulation of leukocytes chemotaxis. These data confirm previous findings in human neonates and provide a model for future studies. Further studies into the biology of the changes in CFI activity in the fetus and the influence of such high risk factors as prematurity, fetal distress or premature rupture of membranes should enhance our understanding of the immunocompromised neonate.

Purification of a Mannan from Candida albicans which Activates Serum Complement

Candida species activate complement by the alternative pathway, induce leukocyte migration and, when applied epicutaneously, cause epidermal microabscesses of neutrophils in man and experimental animals. Complement activation by C. albicans appears to be a property of the cell wall. To biochemically identify the complement-activating constituent(s) of C. albicans, an ethyleneglycol extract of growth phase blastospores was prepared. Acid hydrolysis and neutral sugar analysis revealed mannose (82%), fucose (7%), and glucose (11%). The soluble, mannose-rich cell wall polysaccharide of C. albicans activates serum complement via the alternative pathway, induces neutrophil chemotaxis and is antigenically reactive with antisera to C. albicans. This constituent exhibits in vitro endotoxin-like activity as measured by Limulus lysate gelation, but is nonpyrogenic in rabbits. The extracts produced precipitin lines in double immunodiffusion studies against serum from patients with invasive candidiasis and rabbit antisera to mycelial and blastospore preparations of C. albicans, but not against normal serum. Thus, pathogenic properties and reactive phenomenon of C. albicans are in part attributable to a cell wall polysaccharide, mannan.

Enhancement of Human Neutrophil Bactericidal Activity by Chemotactic Factors

Neutrophils are important effector cells in the defense against microorganisms. They migrate into infected sites and then phagocytose and kill bacteria. Chemotactic factors may be important for initiating neutrophil migration. We investigated whether chemotactic factors might also influence an event subsequent to chemotaxis, namely bacterial killing. It was found that preincubation (20 min at 37 degrees C) of human leukocytes with chemotactic substances such as zymosan-activated serum, a C5a-containing fraction of zymosan-activated serum, N-formyl methionyl phenylalanine or N-formyl methionyl-leucine-phenylalanine, enhanced leukocyte killing of Staphylococcus aureus, Escherichia coli, and Streptococcus faecalis in a dose-dependent fashion. The concentration of chemotactic factor required to enhance killing was similar to that required to induce neutrophil chemotaxis. In addition, zymosan-activated serum, C5a fraction, and the two N-formyl methionyl peptides increased the hexose monophosphate shunt activity of resting and phagocytosing neutrophils by two- to threefold. In contrast, bacterial killing by sodium azide-treated neutrophils and neutrophils from a patient with chronic granulomatous disease was not increased by any chemotactic factor. These findings suggest that chemotactic factors stimulate neutrophil oxygen-dependent microbicidal pathways. These observations illustrate another important contribution of biologically active molecules to effector cell function and host defense.