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Chemotactic factors, which are important in attracting neutrophils to inflammatory sites, have also been shown to stimulate oxidative metabolism, resulting in increased chemiluminescence and release of superoxide anion (O2-). We observed a unique bimodal chemiluminescence pattern upon stimulation with either the complement-derived factor C5a or formyl-methionyl-leucyl-phenylalanine. A sharp peak of activity occurred within 1 to 2 min, and a second more extended peak was seen between 3 and 6 min. Enhancement of both peaks occurred when the cells were pretreated with cytochalasin B. Expression of both peaks was found to be related to cell density, and expression of the second peak was not dependent upon extracellular metabolites released during the first peak. Cells preincubated in luminol and then thoroughly washed responded with only a single peak coincident with the second peak. Together these findings indicate that the first peak is extracellular in origin, whereas the second peak is cell associated. Studies with scavengers of oxygen intermediates and inhibitors of myeloperoxidase for the oxidation of luminol, which may occur in part through the formation of HOCl as well as through a non-HOCl-mediated mechanism. Evidence for a non-HOCl-mediated mechanism comes from experiments in which luminol, myeloperoxidase, and O2- generated by xanthine-xanthine oxidase produce luminescence in the absence of chloride ion. These studies provide further insight into the sequence of events which occur during the stimulation of neutrophils with chemotactic factors and the nature of neutrophil chemiluminescence.
In anaphylactic cutaneous lesions induced by DNP-ascaris extract in the guinea-pig, the time-course of delayed tissue eosinophilia was found to parallel that of the macrophage reaction, reaching its peak in 24 h. Macrophages could be differentiated from lymphocytes by the numerous lysosomal granules which stained for acid phosphatase. Extracts from such skin lesions contained a delayed eosinophil chemotactic factor and two different macrophage chemotactic factors. Most of the delayed eosinophil chemotactic factor was separated from the two macrophage chemotactic factors by gel filtration on Sephadex G-100 and Sephadex G-200 in that order. The eosinophil chemotactic factor after re-chromatography on Sephadex G-I99 showed no or little chemotactic activity for macrophages.
Differences between the conditions for an eosinophil chemotactic factor (ECF) and macrophage chemotactic factor (MCF) production by lymphoid cells of mesenteric lymph nodes and spleen were studied in guinea pigs. If lymphoid cells were washed less than 4 hr after concanavalin A (Con A) stimulation and were cultured for an additional 24 hr, they failed to produce ECF, whereas Con A stimulation for 1 hr before washing was sufficient to stimulate them to produce MCF. Subsequently, it was shown that heat-labile soluble factors (termed ECF-PF) with potentiating activity for ECF production are produced from macrophages by 5 micrograms/ml Con A activation. When ECF-PF were added to the cell culture with 5 micrograms/ml Con A, the lymphoid cells could produce ECF even when they were washed 2 hr after Con A stimulation and were cultured for an additional 24 hr, suggesting that ECF-PF plays a critical role in the early stage of ECF production. The lymphoid cells were also able to produce ECF even when they were cultured with ECF-PF and a suboptimal dose of Con A (1 microgram/ml) for ECF production. Protein synthesis seemed to be essential for ECF-PF production. The ECF-PF activity was associated with two separated molecular fractions with m.w. of about 50,000 to 70,000 and of 10,000 to 20,000. It is thus suggested that ECF is produced from T cells by Con A stimulation under conditions which differ, at least, from those for MCF in the requirement of ECF-PF.
Chemotactic factors, i.e., an N-formyl peptide, C5a, interleukin-8, and leukotriene B4, induced neutrophils to activate mitogen-activated protein (MAP) kinases, as defined by the tyrosine phosphorylation and decrease in electrophoretic mobility of immunodetected 44-, 42-, and 40-kDa proteins. PD 98059, an inhibitor of MAP kinase kinase activation, blocked these changes. The drug likewise blocked neutrophil chemotaxis but did not alter superoxide anion production and paradoxically enhanced degranulation responses to the stimuli. The MAP kinase pathway appears to have a highly selective role in mediating motility but not other cellular responses.
A factor chemotactic for Walker carcinosarcoma and Novikoff hepatoma tumor cells can be generated by incubation of serum with an extract from tumor cells. The chemotactic factor has no activity for neutrophilic leukocytes, and three factors chemotactic for leukocytes are not chemotactic for tumor cells. By the use of complement deficient serums as well as purified complement components, the chemotactic factor for tumor cells has been found to be a fragment of the fifth component (C5) of complement and appears to result from direct interaction of C5 with an enzyme in the extract. The chemotactic factor for tumor cells can be classified as a functionally unique fragment of C5 that may significantly influence the behavior of tumor cells in vivo.
In the previous study, we found an eosinophil chemotactic factor of the parasite (ECF-P) in extracts from the body cavity fluid of the Ascaris species. In this study, the physiochemical characteristics of ECF-P were determined in order to elucidate the relationship between ECF-P and Ascaris antigens. ECF-P was active in both in vivo and in vitro chemotactic assay systems and was found to be heat labile, nondialyzable, and stable to lyophilization. The estimated m.w. was approximately 30,000 daltons. The isoelectric point of ECF-P was 8.5. It was quite different from the Ascaris antigen in the same extract, or various Ascaris allergens that had been reported previously by other researchers. Furthermore, ECF-P did not show any activity in eliciting a guinea pig passive cutaneous anaphylaxis (PCA) reaction. From these experimental results, we concluded that ECF-P is not identical to Ascaris antigens. In addition, we found that the neutrophil chemotactic factor of the parasite (NCF-P), with at least two components, is present in the Ascaris extract, and is also heat labile and resistant to dialysis and lyophilization, but separable from coexistent ECF-P by reason of their different physiochemical properties.
Endotoxic lipopolysaccharide has recently been shown to fix large amounts of the complement components related to the biologic activities mediated by that system. The present study sought to determine whether the generation of chemotactic factor by endotoxin in serum was dependent upon complement system activation. Preheating serum, incubating at 0 degrees C, or incubating in the presence of EDTA, all prevented chemotactic factor generation as well as complement fixation by endotoxin. "Endotoxoids" deficient in complement-firing activity were also deficient in chemotactic factor generation. Chemotactic factor could not be generated by endotoxin in sera of mice congenitally deficient in the C'S component of complement, while chemotactic factor was generated by endotoxin in the sera of coisogenic mice with normal complement levels for that species. The chemotactic factor induced by endotoxin was heat stable and nondialyzable. Molecular sieve chromatography and sucrose density gradient ultracentrifugation demonstrated that the chemotactic factor was a relatively low molecular weight product (15,000-30,000) and as such different from previously scribed C' system-derived chemotactic factors. These experiments demonstrate that generation of chemotactic factor by endotoxin in serum is dependent upon C' system activation involving at least C'5. Furthermore, the relatively low molecular weight of this factor suggests that it might be derived from activation of a single complement component rather than from complexing of multiple complement components.
Cultured bovine aortic endothelial cells secrete a potent migration-stimulating factor for vascular smooth muscle cells (SMCs) and adventitial fibroblasts. Vascular pericytes are 20-fold less responsive, and endothelial cells themselves do not respond at all. Checkerboard analysis of SMC migration in a micro-chemotaxis chamber assay shows that the factor is chemotactic. Chemotactic activity for SMCs and adventitial fibroblasts is specifically inhibited by antibodies against platelet-derived growth factor. Endothelial cells cultured on nitrocellulose filters secrete the platelet-derived growth factor-like factor almost exclusively into the basal compartment. We suggest that this factor plays an important role in the recruitment of vascular wall cells during the morphogenesis of blood vessels and pathological conditions, such as atherosclerosis.
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Idiopathic acquired cold-induced urticaria has provided a model to study release of mast cell-derived chemical mediators into the blood and alterations of neutrophilic leukocyte motility. A factor chemotactic for neutrophilic leukocytes appeared in the circulation after local experimental challenge with ice. After partial purification by Sephadex G-200 gel filtration and by anion and cation exchange chromatography the neutrophil chemotactic activity was excluded on Sepharose 4B gel filtration, indicating a molecular weight in excess of 750,000. On isoelectric focusing it exhibited a neutral isoelectric point. This chemotactic factor showed preferential chemotactic activity for neutrophils and deactivated these cells in vitro and in vivo. HMW-NCF may prove to be a useful marker of mast cell activation and its release may modulate the capacity for motility of neutrophilic leukocytes in humans.
Previous studies have shown that mouse submandibular gland nerve growth factor (NGF) stimulates chemotactic migration of human polymorphonuclear leukocytes in vitro. The results of the present study demonstrate that subdermal injection of NGF in mice also stimulates rapid and marked chemotactic recruitment of leukocytes. This property of NGF is manifest in the nanomolar range of concentrations, it requires the known serine class protease activity of the growth factor, and it does not require participation of the fifth component of complement. Another, as yet unrecognized, C5-independent pathway must be involved. Chemotactic stimulation of cells involved in the early inflammatory response to injury may help to explain earlier observations that NGF can accelerate the rate of contraction of experimentally induced wounds in mice.
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The antitumor effect of PSK, a Coriolus preparation, at a distant site was analyzed with the use of a double grafted tumor system in which male BALB/c mice received simultaneous intradermal inoculations of Meth-A tumor in the right (10(6) cells) and the left (2 x 10(5) cells) flanks and were then injected with PSK in the right tumor on the third day thereafter. The antitumor effect of intratumoral administration of PSK in the right tumor on days 3, 4 and 5 was compared with the effect of surgical resection of the right tumor on day 5. Three out of 8 mice given PSK intratumorally became tumor-free whereas no mouse tumor-free in the left flank was found among the surgically resected mice. As regards sinecomitant immunity, tumor inoculation into the right flank followed by intra-tumoral administration of PSK on days 3 and 5 and surgical excision of the primary tumor on day 6 resulted in complete rejection of a tumor challenge in the left flank on day 21. The combination of presurgical intratumoral injections of PSK (more than 2 times) and postoperative oral administration of PSK appeared to be most effective in eradicating secondary tumors. Isolated TILs (tumor-infiltrating lymphocytes), obtained from the right tumor (treated with PSK) and the left tumor on day 10 in the double grafted tumor system were cultured in RPMI1640 with 10% fetal calf serum for 24 h. The culture supernatants were harvested and tested for the presence of chemotactic activity for neutrophils or macrophages. Significant neutrophil chemotactic factor (NCF) and macrophage chemotactic factor (MCF) activities were detected in the culture media from PSK-treated TILs that had been cultured for 24 h. Neither significant neutrophil nor macrophage chemotactic activity was detected in the media from untreated TILs. NCF and MCF activities were also detected in the culture supernatant from PSK-treated tumor tissue on day 6. PSK-induced NCF in the murine tumor was neutralized by treatment with anti-human IL-8 IgG, and might be murine IL-8-like factor. Therefore, neutrophil and macrophage infiltrations of tumors following intratumoral injections of PSK are probably mediated by inductions of IL-8-like factor and MCF.
Besides its function as a growth factor, the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) "primes" polymorphonuclear leukocytes (PMN) for enhanced biologic responses to a number of secondary stimuli. We examined the effect of priming PMN with GM-CSF on the production of [3H] platelet-activating factor (PAF) from [3H]acetate upon stimulation with the chemotactic factors FMLP and C5a. In PMN stimulated with the individual peptide mediators alone [3H]PAF levels were close to controls, whereas considerable amounts of [3H]PAF are formed after stimulation of PMN which have been preexposed to GM-CSF. The priming effect was concentration and time dependent. It was optimal after a preincubation period of 2 h. A maximum of [3H]PAF accumulation is reached within 2.5 min (C5a) and 5.0 min (FMLP) after activation of GM-CSF-primed PMN. In addition, we show that PAF isolated from PMN preincubated with GM-CSF and triggered with chemotactic factors is able to enhance the respiratory burst in PMN. PAF formed by sequentially activated PMN could contribute to the enhanced oxygen radical production and cytotoxicity in effector cells and play a role in modulating and amplifying inflammatory reactions.
Inflammatory mediators such as the vasoactive and chemotactic factors, which are formed during the activation of serum complement, are extremely potent biologic peptides and are thought to be under the rigid control of specific serum-derived regulators or inactivators, including the anaphylatoxin inactivator (AI) and the chemotactic factor inactivator (CFI). Our understanding of the biologic importance and implication of these regulators in inflammatory reactions is primarily based on in vitro observations, and there is only limited data on their in vivo importance. Previously, alterations in CFI and AI levels were detected in chronic inflammatory disease states, but no data on their activity or role during acute inflammatory reactions have been demonstrated. Here we demonstrate the elevation of serum CFI activity during acute inflammatory reactions in rabbits. Specifically, inflammatory reactions were induced in rabbits by intraperitoneal injections of 0.1% oyster glycogen. Rabbit serum CFI levels rose rapidly over the first 4 hours after glycogen injection, reaching levels 4-8 times higher than the preglycogen serum CFI levels. Additional studies demonstrated that acute elevations of serum CFI levels could also be induced in rabbits by intravenous infusion of activated rabbit serum or C5-derived chemotactic factors. Infusion of saline, albumin, or the synthetic chemotactic peptide f-Met-Leu-Phe did not cause elevation of the serum CFI levels in rabbits. Thus, we take these data to support our hypothesis that 1) CFI is a "hyperacute phase reactant" that is elevated during inflammatory reactions, and 2) that this elevation of serum CFI activity is probably triggered by the appearance of specific C5-derived chemotactic factors within the vasculature. These studies not only provide exciting new insights into the regulatory mechanisms involved in acute inflammatory reactions but suggest that novel approaches to antiinflammatory therapy may be forthcoming.
The mast cell is one of the major effector cells in inflammatory reactions and can be found in most tissues throughout the body. During inflammation, an increase in the number of mast cells can be seen, e.g., in the intraepithelial cell layer after a provoked allergic reaction. Such accumulation probably requires directed migration of mature mast cells or their precursors. To study the migration of human mast cells we used as a model the human mast cell line, HMC-1, and stem cell factor-dependent (also referred to as mast cell growth factor or Kit ligand) cord blood-derived mast cells. The results show that stem cell factor is a potent chemotactic factor for human mast cells in vitro. The chemotactic response to SCF was found to be dose dependent, reaching a maximum at 50 ng/ml. The activity of SCF could be blocked by anti-SCF Abs. We also tested the effect of different intercrines, i.e., IL-8, MIP-1 alpha, MIP-1 beta, RANTES, and MCAF (also referred to as monocyte chemotactic protein 1), on human mast cell migration. Only RANTES was chemotactic for in vitro-developed mast cells. None of the tested intercrines induced migration of HMC-1 cells. For migration, the mast cells were dependent on binding to an extracellular matrix protein. Thus, coating of the filters with fibronectin was required, whereas collagen or laminin did not promote migration. Adhesion of HMC-1 cells to fibronectin could also be shown in an adhesion assay. In addition, expression of receptors for fibronectin could be detected on the surface of the mast cells. These results show that SCF is not only a growth and differentiation factor for human mast cells in vitro but also a potent chemoattractant for such cells.
A chemotactic factor was identified in the supernatants of human large granular lymphocytes (LGL) activated by a glutaraldehyde-fixed NK-sensitive tumor, K562. The factor stimulated migration of human LGL, rat alveolar macrophage (RAM), and human monocytes and neutrophils (PMN). The locomotor response was chemotactic and chemokinetic on the basis of unidirectional migration in concentration gradients. The cell producing the factor was detected exclusively in LGL-rich Percoll fraction coincident with the peak of NK lytic activity and HNK-1+ cells. The monoclonal phenotype of the cell was HNK-1+, partially OKT-11+, OKM-1-, OKT-3-, OKT-4-, and OKT-8-. The factor was released by LGL within 20 min of incubation with Sr++, a cation that is able to induce LGL degranulation. A powerful chemoattractant was also detected in the granules of the rat LGL leukemia, RNK. Chemotactic activity coincided with granule enzyme beta-glucuronidase and cytolysin after RNK nitrogen cavitation and Percoll fractionation of subcellular constituents. The RNK granule chemoattractant induced unidirectional migration of human LGL and was also active against rat alveolar macrophages and human PMN. Anti-RNK granule antibody conjugated to Sepharose 4B was able to deplete the chemotactic activity from both K562-induced LGL supernatants and solubilized RNK granules. These observations indicate that a leukocyte chemotactic factor (NK-LCF) is present in NK cell granules and is probably released after tumor-induced granule exocytosis.