Accumulation of 111In-neutrophils in rabbit skin in allergic and non-allergic inflammatory reactions in vivo. Inhibition by neutrophil pretreatment in vitro with a monoclonal antibody recognizing the CD18 antigen

The mAb 60.3 recognizes the neutrophil CD18 Ag. We have investigated the effect of in vitro pretreatment of radiolabeled neutrophils with mAb 60.3 on their accumulation in vivo. Further, we have compared the in vivo effects of mAb 60.3 with its effects on neutrophil adherence in vitro. Neutrophil accumulation in vivo was measured in response to: 1) exogenous mediators FMLP, C5a des Arg, LTB4 and IL-1; 2) endogenous mediators generated in a non-allergic inflammatory reaction induced by zymosan; and 3) endogenous mediators generated in two allergic inflammatory reactions, a passive cutaneous anaphylactic reaction and a reversed passive Arthus reaction in rabbit skin. Pretreatment of neutrophils with mAb 60.3 inhibited their accumulation in all the responses. The results demonstrate that there is a common mechanism mediating neutrophil accumulation in these inflammatory reactions. Neutrophils pretreated with mAb 60.3 were also unresponsive to chemoattractants in in vitro adherence assays. However, the antibody-treated neutrophils responded normally to FMLP and C5a with respect to granular enzyme release. These results suggest that the basal expression of CD18 Ag is important for the adherence of neutrophils to microvascular endothelial cells stimulated by the local generation, or administration, of chemical mediators in vivo. Despite the fact that mediators such as FMLP can increase CD18 expression in vitro, it appears more likely that such mediators act in vivo by inducing a conformational change in the basally expressed neutrophil adhesive molecules.     

A comparative study of the neutrophil stimulatory activity in vitro and pro-inflammatory properties in vivo of 72 amino acid and 77 amino acid IL-8.

IL-8, a potent neutrophil-activating protein, can be produced by many cell types including monocytes, lymphocytes, fibroblasts, neutrophils, and endothelial cells. Depending on the cell source, the N-terminal amino acid sequence of IL-8 displays heterogeneity that has been shown to confer differences in its neutrophil stimulatory activity in vitro. Despite these observations the relative potency of different IL-8 molecules in vivo is unknown. To address this question we have investigated the biologic activity of the two predominant forms of IL-8, the 72 and the 77 amino acid proteins, in vitro and in vivo. In vitro, human rIL-8(72) and human rIL-8(77) dose dependently induced adherence of rabbit peritoneal neutrophils and human neutrophils to laminin-coated plates and elevated cytoplasmic levels of Ca2+ ([Ca2+]i) in fura-2 loaded neutrophils. In these in vitro assays human rIL-8(72) was more potent than human rIL-8(77) while inducing comparable responses to human rC5a. With respect to enhancing [Ca2+]i, neutrophils desensitized to human rIL-8(72) failed to respond to human rIL-8(77). However, neutrophils fully desensitized to human rIL-8(77) could exhibit a partial response to human rIL-8(72). Further, human rIL-8(72) was approximately 10-fold more effective than human rIL-8(77) in displacing human [125I]rIL-8(72) from rabbit peritoneal neutrophils in a receptor-binding assay. In vivo, intradermally administered human rIL-8(72) and human rIL-8(77) induced 111In-neutrophil accumulation and edema formation in rabbit skin. In contrast to the in vitro studies, the two forms of IL-8 gave identical responses in vivo although they were less potent than human rC5a. Our results demonstrate that, in vitro, human rIL-8(72) is more potent than human rIL-8(77) in stimulating neutrophils. It may be that IL-8)72) has a greater affinity and/or efficacy for the neutrophil IL-8 cell-surface receptors. One possibility for the observation that both forms of IL-8 are equipotent in inducing inflammatory responses in vivo is that the extended form is proteolytically cleaved to the more biologically active IL-8(72).     

Heme induces neutrophil migration and reactive oxygen species generation through signaling pathways characteristic of chemotactic receptors

Hemolysis or extensive cell damage can lead to high concentrations of free heme, causing oxidative stress and inflammation. Considering that heme induces neutrophil chemotaxis, we hypothesize that heme activates a G protein-coupled receptor. Here we show that similar to heme, several heme analogs were able to induce neutrophil migration in vitro and in vivo. Mesoporphyrins, molecules lacking the vinyl groups in their rings, were not chemotactic for neutrophils and selectively inhibited heme-induced migration. Moreover, migration of neutrophils induced by heme was abolished by pretreatment with pertussis toxin, an inhibitor of Galpha inhibitory protein, and with inhibitors of phosphoinositide 3-kinase, phospholipase Cbeta, mitogen-activated protein kinases, or Rho kinase. The induction of reactive oxygen species by heme was dependent of Galpha inhibitory protein and phosphoinositide 3-kinase and partially dependent of phospholipase Cbeta, protein kinase C, mitogen-activated protein kinases, and Rho kinase. Together, our results indicate that heme activates neutrophils through signaling pathways that are characteristic of chemoattractant molecules and suggest that mesoporphyrins might prove valuable in the treatment of the inflammatory consequences of hemorrhagic and hemolytic disorders.     

Inhibition of neutrophil migration by tumor necrosis factor. Ex vivo and in vivo studies in comparison with in vitro effect

Coincubation of neutrophils with TNF inhibited the chemoattractant-directed migration of neutrophils under agarose and enhanced their migration in the multiwell chemotaxis chamber. To assess the physiological significance of these differing in vitro TNF effects, ex vivo and in vivo investigations were performed using animal models. Neutrophils from the peripheral blood of rabbits preadministered systemic TNF showed impaired ability to migrate toward chemoattractants in vitro. In addition, systemic TNF administration suppressed zymosan-activated plasma-induced local accumulation of leukocytes in mouse skin. The results indicate that circulating TNF may act as a suppressor for local inflammatory reaction.     

Inhibition of lymphocyte and neutrophil chemotaxis by pertussis toxin

The cells of the mammalian immune system possess special migratory properties within their in vivo environment, a surveillance characteristic that is thought to be important in the protection of the organism from transformants and exogenous pathogens. Pertussis toxin (PT) has been shown to disrupt the intensity of this process by seriously affecting lymphocyte recirculation in vivo. The mechanisms responsible for this inhibition were investigated by using the in vitro model systems of polymorphonuclear leukocyte and lymphocyte chemotaxis. The type of inhibition that was observed in these in vitro assay systems was quite similar to that observed in vivo, because PT could depress chemotaxis in vitro as well as the accumulation of radiolabeled lymphocytes and neutrophils within a peripheral site of inflammation in vivo. The alterations in neutrophil motility were found to be associated with a stimulus-specific inhibition of the triggering of superoxide anion generation and lysosomal secretion. Some inhibition of neutrophil adherence to plastic surfaces was also observed, most notably after augmentation of adherence with the chemoattractant fMLP. The observed alterations in cellular function after PT treatment occurred in the absence of defects in chemoattractant binding to the neutrophil cell surface, or of membrane potential changes stimulated by ligand binding. The effect of PT in this system was found to be associated with an abnormality in the regulation of intracellular free calcium, suggesting that the substrate for PT in neutrophils is involved in the regulation of calcium ion channels.     

Selective inhibition of human neutrophil chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by sulfones

The therapeutic efficacy of the sulfones, dapsone, and sulfoxone in neutrophilic dermatoses may be related to the effects of these drugs on neutrophil function. Therefore we determined whether neutrophil chemotactic migration to various chemoattractants could be inhibited by sulfones in vitro. The chemotactic responses of human neutrophils from healthy donors were tested by using N-formyl-methionyl-leucyl-phenylalanine (F-met-leu-phe), purified human C5a, and leukocyte-derived chemotactic factor (LDCF). Therapeutic concentrations of sulfones selectively inhibited neutrophil chemotaxis to F-met-leu-phe, but did not affect neutrophil chemotaxis to LDCF or C5a. Inhibition of neutrophil chemotaxis to F-met-leu-phe was induced by both dapsone and sulfoxone at a concentration of 10 micrograms/ml without affecting random migration, and the inhibition was reversed by washing the neutrophils. When dapsone- and sulfoxone-treated neutrophils (100 micrograms/ml) were stimulated with F-met-leu-phe, neutrophil superoxide generation was not inhibited. Sulfapyridine (10 micrograms/ml) also selectively inhibited neutrophil chemotaxis to F-met-leu-phe; however, sulfamethoxazole and sulfisoxazole did not affect chemotaxis. The inhibitory effects of dapsone, sulfoxone, and sulfapyridine could not be demonstrated with granulocytes from rabbits or guinea pigs nor with human monocytes. Experiments with radiolabeled dapsone showed rapid, nonspecific, and reversible binding of dapsone to human neutrophils. These data suggest that a mechanism of action of sulfones in neutrophilic dermatoses may be a selective inhibition of neutrophil migration to as yet undefined chemoattractants in the skin.     

Mediators and regulation of neutrophil accumulation in inflammatory responses in lung: insights from the IgG immune complex model

Neutrophil trafficking in lung involves transendothelial migration, migration in tissue interstitium, and transepithelial migration. In a rat model of IgG immune complex-induced lung injury, it was demonstrated that neutrophil emigration involves regulatory mechanisms including complement activation, cytokine regulation, chemokine production, activation of adhesion molecules, and their respective counter receptors. The process is presumably initiated and modulated by the production of early response cytokines and chemokines from lung cells, especially from alveolar macrophages. TNF-alpha and IL-1 up-regulate intracellular adhesion molecule-1 (ICAM-1) and E-selectin, setting the stage for neutrophil migration through endothelium. The CXC chemokines, such as macrophage inflammatory protein (MIP)-2 and cytokine-inducible neutrophil chemoattractant (CINC), constitute chemokine gradient to orchestrate neutrophil migration in lung. Complement activation induced by IgG immune complex deposition is another important event leading to neutrophil accumulation in lung. Complement activation product C5a not only plays an important role in chemoattracting neutrophils into lung, but regulates adhesion molecules, chemokines, and cytokines expression. In addition, oxidative stress may regulate neutrophil accumulation in lung by modulation of adhesion molecule activation and chemokine production. In this review, we focus on the current knowledge of the mechanisms leading to accumulation of neutrophils during acute lung injury.     

Flotillin microdomains interact with the cortical cytoskeleton to control uropod formation and neutrophil recruitment

We studied the function of plasma membrane microdomains defined by the proteins flotillin 1 and flotillin 2 in uropod formation and neutrophil chemotaxis. Flotillins become concentrated in the uropod of neutrophils after exposure to chemoattractants such as N-formyl-Met-Leu-Phe (fMLP). Here, we show that mice lacking flotillin 1 do not have flotillin microdomains, and that recruitment of neutrophils toward fMLP in vivo is reduced in these mice. Ex vivo, migration of neutrophils through a resistive matrix is reduced in the absence of flotillin microdomains, but the machinery required for sensing chemoattractant functions normally. Flotillin microdomains specifically associate with myosin IIa, and spectrins. Both uropod formation and myosin IIa activity are compromised in flotillin 1 knockout neutrophils. We conclude that the association between flotillin microdomains and cortical cytoskeleton has important functions during neutrophil migration, in uropod formation, and in the regulation of myosin IIa.     

Role of 5-lipoxygenase products in the local accumulation of neutrophils in dermal inflammation in the rabbit.

Studies were undertaken to define the role of 5-lipoxygenase (5-LO) products and, in particular, of leukotriene (LT) B4 in the polymorphonuclear leukocyte (PMN) emigration process using a rabbit model of dermal inflammation. Our results show that i.v. administration to rabbits of MK-0591, a compound that inhibits LT biosynthesis in blood and tissues when administered in vivo, significantly reduced 51Cr-labeled PMN accumulation in response to intradermally injected chemotactic agonists, including IL-8, FMLP, C5a, and LTB4 itself. In addition, pretreatment of the labeled PMN with MK-0591 ex vivo before their injection in recipient animals was equally effective in reducing 51Cr-labeled PMN emigration to dermal inflammatory sites. These results support a role for de novo synthesis of 5-LO metabolites by PMN for their chemotactic response to inflammatory mediators. Other studies demonstrated that elevated intravascular concentration of LTB4 interferes with PMN extravasation inasmuch as a continuous i.v. infusion of LTB4, in the range of 5-300 ng/min/kg, dose-dependently inhibited extravascular PMN accumulation to acute inflammatory skin sites elicited by the chemoattractants LTB4, FMLP, C5a, and IL-8 and by TNF-alpha, IL-1beta, and LPS; such phenomena may constitute a natural protective mechanism from massive tissue invasion by activated PMN in specific pathologic conditions such as ischemia (and reperfusion). These studies demonstrate additional functions of 5-LO products in the regulation of PMN trafficking, distinct from the well-characterized chemotactic activity of LTB4 present in the extravascular compartment.     

Characterization of a receptor for human monocyte-derived neutrophil chemotactic factor/interleukin-8

Monocyte-derived neutrophil chemotactic factor/interleukin-8 (MDNCF/IL-8) is an 8,000-dalton protein produced by monocytes which exhibits activity as a chemoattractant for neutrophils with maximal activity achieved at a concentration of 50 ng/ml. This polypeptide has been iodinated by chloramine-T methodology (350 Ci/mM), and specific receptors for MDNCF/IL-8 have been detected on human neutrophils, U937 cells, THP-1 cells, and dimethyl sulfoxide-differentiated HL-60 cells. The binding of MDNCF/IL-8 to human neutrophils is not inhibited by interleukin-1 alpha, tumor necrosis factor-alpha, insulin, or epidermal growth factor. In addition, chemoattractants such as C5a, fMet-Leu-Phe, leukotriene B4, and platelet-activating factor fail to inhibit binding, suggesting that MDNCF/IL-8 utilizes a unique receptor. The receptor for MDNCF/IL-8 is apparently glycosylated since ligand binding is inhibited by the presence of wheat germ agglutinin, a lectin with a binding specificity for N-acetylglucosamine and neuraminic acid. Steady state binding experiments indicate Kd values of 4 and 0.5 nM and receptor numbers of 75,000 and 7,400 for human neutrophils and differentiated HL-60 cells, respectively. 125I-MDNCF/IL-8 bound to human neutrophils is rapidly internalized and subsequently released from cells as trichloroacetic acid-soluble radioactivity. Affinity labeling experiments suggest that the human neutrophil MDNCF/IL-8 receptor exhibits a mass of approximately 58,000 daltons.     

A novel neutrophil chemoattractant generated during an inflammatory reaction in the rabbit peritoneal cavity in vivo. Purification, partial amino acid sequence and structural relationship to interleukin 8.

An inflammatory reaction was induced in vivo by injection of zymosan into the peritoneal cavity of the rabbit. The inflammatory exudate was found to contain oedema-inducing and neutrophil chemoattractant activity when assayed in rabbit skin in vivo, using 125I-albumin and 111In-neutrophils. This activity was additional to that of complement fragment C5a, which was removed by an affinity gel. Two chemoattractants were isolated by cation-exchange, gel-filtration and reversed-phase h.p.l.c. One of these, which ran as a single band of 6-8 kDa on SDS/PAGE, was subjected to N-terminal sequence analysis without reduction and alkylation of cysteine residues. Positive identification of 28 of the first 31 amino acids revealed a rabbit homologue of interleukin-8 (75% sequence identity with human interleukin-8). The demonstration of interleukin-8 as a major neutrophil chemoattractant in an inflammatory reaction in vivo provides the basis for further investigations into the role of this cytokine in the inflammatory process.     

12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid is a more potent neutrophil chemoattractant than the 12(R) epimer in the rat cornea

12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(R)-HETE] is reported to be more potent than its epimer 12(S)-HETE as a chemoattractant for human neutrophils in vitro and following topical application to the skin. To assess the in vivo neutrophil chemoattractant potencies of 12(S)-HETE and 12(R)-HETE in the rat, we injected 1 microgram, 5 micrograms, or 10 micrograms of these eicosanoids into the corneal stroma. Rats were killed 12-15 hours after injection, and the number of neutrophils in the stroma was counted in a histological section of the cornea including the injection site. The number of neutrophils was significantly increased in corneas injected with 5 micrograms (+103% of control) or 10 micrograms (+456% of control) of 12(S)-HETE and in those injected with 10 micrograms of 12(R)-HETE (+111% of control). The neutrophilic infiltrate in corneas injected with 1 microgram or 5 micrograms of 12(S)-HETE was not significantly different from that in corneas injected with 1 microgram of leukotriene B4. The data for the 10 micrograms injections indicate that 12(S)-HETE is a more potent neutrophil chemoattractant than 12(R)-HETE in the rat cornea. Our results suggest that species or tissue specificity may determine the relative potencies of 12-HETE epimers as chemoattractants for neutrophils, and that 12(S)-HETE may be an important inflammatory mediator in the rat cornea.     

Herpesvirus chemokine-binding glycoprotein G (gG) efficiently inhibits neutrophil chemotaxis in vitro and in vivo

Glycoprotein G (gG) of alphaherpesviruses has been described to function as a viral chemokine-binding protein (vCKBP). More recently, mutant viruses devoid of gG have been shown to result in increased virulence, but it remained unclear whether the potential of gG to serve as a vCKBP is responsible for this observation. In this study, we used equine herpesvirus type 1 (EHV-1) as a model to study the pathophysiological importance of vCKBP activity. First, in vitro chemotaxis assays studying migration of immune cells, an important function of chemokines, were established. In such assays, supernatants of EHV-1-infected cells significantly inhibited IL-8-induced chemotaxis of equine neutrophils. Identification of gG as the responsible vCKBP was achieved by repeating similar experiments with supernatants from cells infected with a gG-negative mutant, which were unable to alter IL-8-induced equine neutrophil migration. Furthermore, rEHV-1 gG was able to significantly reduce neutrophil migration, establishing gG as a bona fide vCKBP. Second, and importantly, in vivo analyses in a murine model of EHV-1 infection showed that neutrophil migration in the target organ lung was significantly reduced in the presence of gG. In summary, we demonstrate for the first time that EHV-1 gG not only binds to chemokines but is also capable of inhibiting their chemotactic function both in vitro and in vivo, thereby contributing to viral pathogenesis and virulence.     

LTB4 is a signal-relay molecule during neutrophil chemotaxis

Neutrophil recruitment to inflammation sites purportedly depends on sequential waves of chemoattractants. Current models propose that leukotriene B(4) (LTB(4)), a secondary chemoattractant secreted by neutrophils in response to primary chemoattractants such as formyl peptides, is important in initiating the inflammation process. In this study we demonstrate that LTB(4) plays a central role in neutrophil activation and migration to formyl peptides. We show that LTB(4) production dramatically amplifies formyl peptide-mediated neutrophil polarization and chemotaxis by regulating specific signaling pathways acting upstream of actin polymerization and MyoII phosphorylation. Importantly, by analyzing the migration of neutrophils isolated from wild-type mice and mice lacking the formyl peptide receptor 1, we demonstrate that LTB(4) acts as a signal to relay information from cell to cell over long distances. Together, our findings imply that LTB(4) is a signal-relay molecule that exquisitely regulates neutrophil chemotaxis to formyl peptides, which are produced at the core of inflammation sites.     

Neutrophil migration induced by IL-8-activated mast cells is mediated by CINC-1

The aim of this study was to characterize the mediators released by mast cells responsible for IL-8-induced neutrophil migration. It was observed that IL-8 induces a dose-dependent neutrophil migration into peritoneal cavity of rats, but not into air-pouch cavity in which resident mast cells are not present. The transference of peritoneal mast cells to the air-pouch renders this cavity responsive to IL-8. The neutrophil migration induced by IL-8 into the peritoneal cavity was not observed when the peritoneal-resident mast cells were depleted by compound 48/80 or distilled water treatment. Confirming the importance of mast cells, IL-8-stimulated mast cells supernatant induced significant neutrophil migration when injected into peritoneal and air-pouch cavities. The IL-8-induced neutrophil migration was observed not to be dependent on LTB(4), prostaglandins or TNF-alpha, since MK886, indomethacin or thalidomide were unable to block the IL-8-induced neutrophil accumulation 'in vivo' or the release of neutrophil chemotactic factor "in vitro" by IL-8-stimulated mast cells. However, dexamethasone, an inhibitor of the synthesis of pro-inflammatory cytokines, blocked the neutrophil migration induced by IL-8 "in vivo" and also inhibited the release of the neutrophil chemotactic factor by IL-8-stimulated mast cells. Moreover, the incubation of IL-8-stimulated mast cells supernatant with antibody against cytokine-induced neutrophil chemoattractant 1 (CINC-1), but not against TNF-alpha or IL-1beta, inhibited its neutrophil chemotactic activity. Furthermore, we found a significant amount of CINC-1 in this supernatant. In conclusion, we demonstrated that the neutrophil migration induced by IL-8 is dependent on CINC-1 release from mast cells.     

The major outer sheath protein of Treponema denticola selectively inhibits Rac1 activation in murine neutrophils

Treponema denticola major outer sheath protein (Msp) inhibits neutrophil chemotaxis in vitro , but key regulatory mechanisms have not been identified. Because the Rac small GTPases regulate directional migration in response to chemoattractants, the objective was to analyse the effects of Msp on formyl -methionyl-leucyl-phenylalanine (fMLP)-mediated neutrophil polarization and Rac activation in murine neutrophils. Msp pretreatment of neutrophils inhibited both polarization and chemotactic migration in response to fMLP. Activation of small GTPases was measured by p21 binding domain (PBD) pulldown assays, followed by Western analysis, using monoclonal anti-Rac1, anti-Rac2, anti-cdc42 and anti-RhoA antibodies. Enriched native Msp selectively inhibited fMLP-stimulated Rac1 activation in a concentration-dependent manner, but did not affect Rac2, cdc42 or RhoA activation. Murine neutrophils transfected with vectors expressing fluorescent probes PAK-PBD-YFP and PH-AKT-RFP were used to determine the effects of Msp on the localization of activated Rac and PI3 kinase products. Real-time confocal images showed that Msp inhibited the polarized accumulation of activated Rac and PI3-kinase products upon exposure to fMLP. The findings indicate that T. denticola Msp inhibition of neutrophil polarity may be due to the selective suppression of the Rac1 pathway.     

Modulation of IL-1, tumor necrosis factor, and C5a-mediated murine neutrophil migration by alpha-melanocyte-stimulating hormone

This study was designed to examine the effects of i.p.-injected alpha-melanocyte stimulating hormone (MSH) on murine neutrophil migration into subcutaneously implanted sponges in response to IL-1-alpha, TNF-alpha, and C5a. The results show that as little as 0.1 ml of 5 x 10(-7) M MSH injected i.p. significantly blocked the accumulation of neutrophils in sponges in response to IL-1. This action of MSH was dose dependent, reversible, and was maximally effective if MSH was given at the same time as the injection of IL-1. This effect of MSH was not restricted to IL-1-induced neutrophil emigration, because MSH also antagonized the accumulation of neutrophils in response to both TNF and C5a. The proopiomelanocortin-derived peptide ACTH which contains the MSH sequence also significantly reduced neutrophil accumulation in response to IL-1, although less effectively than MSH. Similar studies with beta-endorphin showed that it had no effect on neutrophil accumulation in this system. The direct injection of MSH, beta-endorphin and ACTH into sponges or i.p. did not stimulate a neutrophil emigration and eliminated the possibility that MSH or ACTH suppressed the neutrophil influx in response to IL-1, TNF, or C5a by competing for circulating neutrophils. The action of MSH on IL-1, TNF, and C5a-induced neutrophil emigration suggests that this peptide may be an important regulator of the inflammatory response.     

Inhibition of IL-8-induced W3/25+ (CD4+) T lymphocyte recruitment into subcutaneous tissues of rats by selective depletion of in vivo neutrophils with a monoclonal antibody

IL-8 recruits both neutrophils and lymphocytes in vitro and in vivo. To elucidate the mechanisms of lymphocyte recruitment in vivo by IL-8, we examined the role of neutrophil infiltration through selective depletion of circulating neutrophils using a mAb, RP-3. Selective depletion of neutrophils inhibited the IL-8 induced in vivo migration of the W3/25+ (CD4+) T cell subset but did not inhibit that of the MRC-OX8+ (CD8+) subset. These results suggest that CD4+ T cell migration into IL-8-injected s.c. tissues depends on the prior infiltration of neutrophils chemoattracted directly by IL-8.     

Biological characterization of purified macrophage-derived neutrophil chemotactic factor

We have recently described the purification of a 54 kDa acidic protein, identified as macrophage-derived neutrophil chemotactic factor (MNCF). This protein causes in vitro chemotaxis as well as in vivo neutrophil migration even in animals treated with dexamethasone. This in vivo chemotactic activity of MNCF in animals pretreated with dexamethasone is an uncommon characteristic which discriminates MNCF from known chemotactic cytokines. MNCF is released in the supernatant by macrophage monolayers stimulated with lipopolysaccharide (LPS). In the present study, we describe some biological characteristics of homogenous purified MNCF. When assayed in vitro, MNCF gave a bell-shaped dose-response curve. This in vitro activity was shown to be caused by haptotaxis. Unlike N-formyl-methionylleucyl- phenylalanine (FMLP) or interleukin 8 (IL-8), the chemotactic activity of MNCF in vivo and in vitro, was inhibited by preincubation with D-galactose but not with D-mannose. In contrast with IL-8, MNCF did not bind to heparin and antiserum against IL-8 was ineffective in inhibiting its chemotactic activity. These data indicate that MNCF induces neutrophil migration through a carbohydrate recognition property, but by a mechanism different from that of the known chemokines. It is suggested that MNCF may be an important mediator in the recruitment of neutrophils via the formation of a substrate bound chemotactic gradient (haptotaxis) in the inflamed tissues.     

A sensitive chemiluminescence assay for pertussis toxin and for evaluation of cell-free pertussis toxoids

Pertussis toxin (PT) inhibited luminol-enhanced chemiluminescence induced in rabbit peritoneal neutrophils by N'-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) at doses as low as 0.8 ng.ml-1, even in the presence of a 10-fold higher concentration of filamentous haemagglutinin (FHA). A cell-free extract of Bordetella pertussis, containing predominantly PT and FHA, suppressed the neutrophil response to fMLP. After toxoiding with carbodiimide, the inhibitory activity of the extract was abolished and an enhancement of neutrophil chemiluminescence was observed due to FHA activity. Abrogation of the chemiluminescent response of neutrophils to fMLP is proposed as a sensitive, in vitro assay for pT, and may be useful for monitoring the residual toxin activity in pertussis toxoids and for determining the anti-toxic effects of anti-PT antibodies.