Increase In Chemotactic Activity Research Articles

Haploinsufficiency of the folliculin gene leads to impaired functions of lung fibroblasts in patients with Birt-Hogg-Dubé syndrome.

Birt–Hogg–Dubé syndrome (BHDS) is an autosomal dominant inherited disorder caused by germline mutations in the FLCN gene, and characterized by skin fibrofolliculomas, multiple lung cysts, spontaneous pneumothorax, and renal neoplasms. Pulmonary manifestations frequently develop earlier than other organ involvements, prompting a diagnosis of BHDS. However, the mechanism of lung cyst formation and pathogenesis of pneumothorax have not yet been clarified. Fibroblasts were isolated from lung tissues obtained from patients with BHDS (n = 12) and lung cancer (n = 10) as controls. The functional abilities of these lung fibroblasts were evaluated by the tests for chemotaxis to fibronectin and three‐dimensional (3‐D) gel contraction. Fibroblasts from BHDS patients showed diminished chemotaxis as compared with fibroblasts from controls. Expression of fibronectin and TGF‐β1 was significantly reduced in BHDS fibroblasts when assessed by qPCR. Addition of TGF‐β1 in culture medium of BHDS lung fibroblasts significantly restored these cells' abilities of chemotaxis and gel contraction. Human fetal lung fibroblasts (HFL‐1) exhibited reduced chemotaxis and 3‐D gel contraction when FLCN expression was knocked down. To the contrary, a significant increase in chemotactic activity toward to fibronectin was demonstrated when wild‐type FLCN was overexpressed, whereas transduction of mutant FLCN showed no effect on chemotaxis. Our results suggest that FLCN is associated with chemotaxis in lung fibroblasts. Together with reduced TGF‐β1 expression by BHDS lung fibroblasts, a state of FLCN haploinsufficiency may cause lung fibroblast dysfunction, thereby impairing tissue repair. These may reveal one mechanism of lung cyst formation and pneumothorax in BHDS patients.

Induction of Macrophage Chemotaxis by Aortic Extracts from Patients with Marfan Syndrome Is Related to Elastin Binding Protein

Marfan syndrome is an autosomal dominantly inherited disorder of connective tissue with prominent skeletal, ocular, and cardiovascular manifestations. Aortic aneurysm and dissection are the major determinants of premature death in untreated patients. In previous work, we showed that extracts of aortic tissues from the mgR mouse model of Marfan syndrome showed increased chemotactic stimulatory activity related to the elastin-binding protein. Aortic samples were collected from 6 patients with Marfan syndrome and 8 with isolated aneurysms of the ascending aorta. Control samples were obtained from 11 organ donors without known vascular or connective tissue diseases. Soluble proteins extracted from the aortic samples of the two patient groups were compared against buffer controls and against the aortic samples from controls with respect to the ability to induce macrophage chemotaxis as measured using a modified Boyden chamber, as well as the reactivity to a monoclonal antibody BA4 against bioactive elastin peptides using ELISA. Samples from Marfan patients displayed a statistically significant increase in chemotactic inductive activity compared to control samples. Additionally, reactivity to BA4 was significantly increased. Similar statistically significant increases were identified for the samples from patients with idiopathic thoracic aortic aneurysm. There was a significant correlation between the chemotactic index and BA4 reactivity, and the increases in chemotactic activity of extracts from Marfan patients could be inhibited by pretreatment with lactose, VGVAPG peptides, or BA4, which indicates the involvement of EBP in mediating the effects. Our results demonstrate that aortic extracts of patients with Marfan syndrome can elicit macrophage chemotaxis, similar to our previous study on aortic extracts of the mgR mouse model of Marfan syndrome (Guo et al., Circulation 2006; 114:1855-62).

In vitro Effect of α-tocopherol and Ascorbic Acid Supplementation on Immunological Indicators in Bovine Leukocytes following Transportation

The aim of the present study was to evaluate the effect of two different antioxidants, α-tocopherol and ascorbic acid, on the chemotactic activity and susceptibility to Mannheimia haemolytica leukotoxin (Lkt) of white blood cells obtained from calves exposed to transport in vitro. Both antioxidants used were found to affect the chemotactic activity of white blood cells. Supplementation of α-tocopherol at a concentration of 0.2 mg/ml and 0.5 mg/ml induced significant (P ⪬ 0.05) increase of chemotactic activity in white blood cells compared to control. In case of ascorbic acid supplementation the highest chemotactic activity was observed at a concentration of 0.5 mg/ml. The results obtained in microtitration (MTT) assay also suggest that high concentrations of α-tocopherol (0.5 mg/ml) play a role in protecting bovine leukocytes against the cytotoxic effect of Lkt, the susceptibility of the leukocytes on M. haemolytica Lkt was lower in comparison to control about 6%. The antioxidants used in this study have been found to have a protective effect on cells participating in the immune response to M. haemolytica. This particularly applies to high concentrations of α-tocopherol. The clinical aspect of these results may lie in protection of leukocytes exposed to leukotoxin of M. haemolytica. It should be emphasized that the results provide only partial information on the effect of the antioxidants studied on cells participating in an organism’s defence mechanisms. For an evaluation that would enable proper prevention of bovine respiratory diseases, research should be continued and expanded to include analysis of cellular structures.

Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (TAFIa) and Platelets.

Chemerin is a potent chemoattractant for cells that express the serpentine receptor CMKLR1 (chemokine-like receptor 1), such as plasmacytoid dendritic cells and tissue macrophages. Chemerin circulates in the blood in a relatively inactive form (prochemerin). Its chemotactic activity is unleashed following proteolytic cleavage of carboxyl-terminal amino acids by serine proteases including plasmin, factor XIIa and VIIa. Recruitment of plasmacytoid dendritic cells and macrophages by chemerin may play a role in local tissue immune and inflammatory responses. The shortest bioactive chemerin peptide NH2-YFPGQFAFS-COOH (9mer) is present in the carboxyl-terminal domain. In this work, we show that plasma carboxypeptidase N (CPN) and B (CPB or activated thrombin-activatable fibrinolysis inhibitor (TAFIa)) (30 nM) remove the C-terminal lysine (K) from YFPGQFAFSK (10mer) (200nM) and enhance the migration of CMKLR1-transfected cells by ∼16-fold. To investigate if sequential proteolysis by plasmin and carboxypeptidases can modulate the activity of chemerin peptides, we generated the carboxyl-terminal 15mer of prochemerin, NH2-YFPGQFAFSKALPRS-COOH. Plasmin cleavage (1 μM) generated a 10mer, which was further processed to 9mer by CPN or CPB (30 nM) cleavage. These sequential cleavages were paralleled by corresponding increases in chemotactic activity. At concentrations as high as 1 μM the 15 mer did not induce chemotaxis; after plasmin cleavage and conversion to 10mer, however, significant chemotactic activity was demonstrated. Treatment with CPN or CPB further enhanced this chemotactic activity. We observed a similar enhancement in bioactivity following sequential plasmin/CPN-or-CPB cleavage of full-length prochemerin. Endogenous plasma CPN supports the activation (∼2.5 fold increase in bioactivity) of plasmin-cleaved prochemerin (0.2 nM). This activation was blocked by incubating plasma with MGTA (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid), a specific CPN inhibitor. In addition, we show that platelets are a rich source of chemerin (20–30 ng of chemerin/5x108 platelets). Chemerin released from activated platelets triggers CMKLR1-transfectant chemotaxis, which was blocked by anti-chemerin antibodies. Thus, the circulating humoral factors reported here (platelets, serine proteases, and carboxypeptidases) may contribute to the regulation of chemerin bioactivity in vivo and therefore play a critical role in CMKLR1-mediated immune responses.

Altered chemotactic behavior of crevicular PMNs in different forms of periodontitis.

Polymorphonuclear neutrophils (PMN) are the predominant host defence cells in the gingival sulcus. Previous work demonstrates that the in vitro phagocytosis of crevicular cells in localised early onset periodontitis (LEOP) and generalised early onset periodontitis (GEOP) lesions is diminished. The present study extends this work by characterizing the chemotaxis function of crevicular fluid (CF) PMNs in various forms of periodontitis. We investigated 7 patients with LEOP, 11 patients with GEOP, 12 patients with adult periodontitis (AP) and 2 age- and sex-matched healthy control groups. The two deepest sites of each quadrant in test and control subjects were selected for crevicular sampling. Chemotaxis was performed in a micro chamber (moist atmosphere, 5% CO2, 37 degrees C, 30 min) using N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 x 10(-7) mol FMLP/l) as a chemoattractant. The total chemotaxis was defined as the difference between the number of cells migrating towards FMLP minus the number of cells migrating towards PBS, counted in 20 randomly selected fields. Membranes were examined microscopically at 400 x magnification. The chemotactic activity in the adult periodontitis group was significantly higher compared to the age-related control group. However, we found a statistically significant reduction of chemotactic activity in LEOP and GEOP patients compared to the controls. These results indicate an increase of chemotactic activity from CF-PMN in patients with adult periodontitis, but on the other hand, a significant reduction of chemotactic responsiveness of these cells in LEOP and GEOP lesions.

Local production of interleukin-5 by T lymphocytes is associated with recruitment of eosinophils in patients with eosinophilic granuloma of the soft tissue.

Eosinophilic granuloma of the soft tissue (EOSG) is a rare disease of unknown cause. Since the in vivo mechanism of eosinophilia remains unclear, the present study was performed to investigate the mechanism of the infiltration of eosinophils into the granuloma tissue. Immunohistochemical techniques and an eosinophil chemotactic assay were used in analysis. Peripheral blood eosinophils obtained from one patient showed an increased chemotactic response against tissue extract that was inhibited by pretreatment with anti-IL-5 antibodies. Eosinophils obtained from the peripheral blood of patients with EOSG showed a significant increase in chemotactic activity toward 10(-9) M recombinant human (rh) IL-5 versus that of healthy individuals, whereas eosinophils from granuloma tissue showed no chemotactic response toward rhIL-5, indicating that IL-5 may deactivate the eosinophils. Immunohistochemical studies showed that CD4+ cells were predominantly found in the extrafollicular region, along with interleukin-5+ (IL-5) cells. Staining of the adjacent 3-micrometers sections for CD3, eosinophils, and IL-5 revealed that most of the IL-5 immunoreactive CD3+ cells exhibited cytoplasmic staining. Conversely, 97% of IL-5+ eosinophils were stained peripherally in a ring-like manner, suggesting that IL-5 was bound to its cell surface receptor on the eosinophil. IL-5 mRNA expression was detectable in the CD3+T lymphocytes but not in eosinophils from granuloma tissue. These findings suggest that locally produced IL-5 from T lymphocytes may enhance the infiltration of eosinophils into the eosinophilic granuloma.

Cytokine-induced neutrophil chemoattractant mediates neutrophilic alveolitis in rats: association with nuclear factor kappa B activation.

Cytokine-induced neutrophil chemoattractant (CINC) is a rat cytokine with structural and functional homology to human interleukin-8 (IL-8) and melanoma growth-stimulatory activity (MGSA/gro). We investigated the relationship between CINC and the production of chemotactic activity for neutrophils by rat alveolar macrophages after in vitro and in vivo treatment with endotoxin. After in vitro treatment with endotoxin, the chemotactic bioactivity produced by alveolar macrophages increased in a time- and dose-dependent manner. This increase in chemotactic activity was closely associated with increased levels of steady-state CINC mRNA. About 50% of the chemotactic activity was blocked by treatment with neutralizing concentrations of anti-CINC antibodies. We then evaluated the role of CINC in vivo in the development of neutrophilic alveolitis in rats, which results from a single intraperitoneal injection of endotoxin. In this model, peak numbers of neutrophils are recovered in lung lavage fluid 24 h after endotoxin injection. Steady-state CINC mRNA levels in the lung peaked 2 h after endotoxin injection. Many cytokines whose transcription is induced during sepsis, including IL-8 and MGSA/gro, are thought to be transcriptionally regulated by nuclear factor kappa B (NF-kappa B). The CINC gene contains a binding site in the promoter region for NF-kB. Therefore, we sought to determine whether NF-kappa B binding to the CINC NK-kappa B motif was increased in nuclear extracts from rat lung lavage cells after exposure to endotoxin using gel mobility shift assays. Increased nuclear NF-kappa B binding activity was detected 2.5 h after in vivo treatment with endotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement activation by cigarette smoke

Lung disease secondary to cigarette smoking is associated with an influx of neutrophils and monocytes into the lower respiratory tract. To determine whether cigarette smoke can generate chemotactic activity, human serum was exposed to cigarette smoke and evaluated for neutrophil and monocyte chemotactic activity. Serum exposed to cigarette smoke attracted significantly greater numbers of neutrophils and monocytes compared with normal human serum exposed to air (P less than 0.01, both comparisons). The increase in chemotactic activity was partially attenuated by EDTA but not by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and MgCl2 (P greater than 0.05, both comparisons), suggesting activation of the alternate complement pathway. To evaluate the capacity of cigarette smoke to activate the complement system, smoke-exposed serum was evaluated for cleavage of properdin factor B and C3 using immunoelectrophoresis and for C5a using a radioimmunoassay. Cleavage of properdin factor B and C3 was observed in the smoke-exposed serum and C5a was detected in the smoke-exposed serum (112 +/- 31 ng/ml). These data suggest that complement activation may play a role in directing the influx of neutrophils and monocytes into the lungs of cigarette smokers.

Characterization of Methylated Bovine Serum Albumin-Induced Allergic Inflammation in Rats

An air pouch type allergic inflammation in rats was induced using an insoluble cationic protein, methylated bovine serum albumin (MeBSA), as an antigen. Changes in vascular permeability, local tissue edema, histamine contents in the pouch fluid, and number of infiltrated leukocytes and chemotactic activity in the pouch fluid were analyzed during an 8-hour period after injecting the antigen solution into the air pouch of the immunized and nonimmunized rats. Vascular permeability during the first 30-min interval in the immunized rats was higher than that in the nonimmunized rats, reflecting a higher histamine level in the pouch fluid. However, both the increase in vascular permeability and histamine level in the immunized rats in this period were much lower than those induced by a soluble, noncationic antigen, azobenzenearsonate-conjugated acetyl bovine serum albumin. In the MeBSA-induced allergic inflammation model, a second peak of vascular permeability was induced at 2 h, and local tissue edema formation became apparent at 2 h, reaching a plateau at 4 h. A prominent increase in leukocyte infiltration, especially neutrophils, into the pouch fluid was induced at 4 h in accordance with an increase in chemotactic activity in the pouch fluid. These observations indicate that the acute phase of MeBSA-induced allergic inflammation is characterized by a weak anaphylactic response and a prominent neutrophil infiltration.

Secretion by human fibroblasts of monocyte chemoattractant protein-1, the product of gene JE.

We recently purified human monocyte chemoattractant protein-1 (MCP-1) from culture fluids of either human glioma cell lines or mitogen-stimulated human peripheral blood mononuclear leukocytes. It has now been shown that MCP-1 is the product of the gene JE, which was first recognized by its expression in fibroblasts stimulated with platelet-derived growth factor (PDGF). We therefore studied secretion of MCP-1 by three human fibroblast cell lines. Monocyte chemotactic activity was found in culture fluids of all three lines after growth to confluence in DMEM-10% FCS, and the amounts secreted per cell were comparable for the three lines. The MRC-5 line was chosen for further study. Monocyte chemotactic activity secretion by confluent MRC-5 cultures continued after a switch to serum-free medium and was not inhibited by anti-PDGF antibody, indicating that secretion may not have been caused by autocrine release of PDGF. When concentrated serum-free MRC-5 culture fluid was injected into an HPLC gel filtration column, only one chemotactic activity peak was observed, which was in the same location as glioma-derived MCP-1. The activity was completely absorbed out by an anti-MCP-1 affinity column, which indicates that all the chemotactic activity in MRC-5 culture fluid was accounted for by MCP-1. PDGF caused a marked increase in chemotactic activity over that found in serum-free culture fluid of MRC-5 or 501T cells. Immunoprecipitation by anti-human MCP-1 showed two bands, corresponding to the two forms of MCP-1 previously described (MCP-1 alpha and beta); and the amounts increased in response to PDGF stimulation. Thus, the reported increase in human fibroblast JE mRNA in response to PDGF-containing serum stimulation is reflected in increased secretion of the MCP-1 gene product.

Inflammatory Mediators in Inflammatory Bowel Disease

Tissue responses to an inflammatory stimulus (such as vasodilation, plasma exudation invasion and activation of inflammatory cells) are elicited by locally synthesized chemical mediators. Inhibition of biosynthesis and/or antagonism of action of these mediators is an important target of drug therapy, particularly when the cause of the disease is unknown. Recent investigations have revealed that the mucosa of inflammatory bowel disease (IBD) patients synthesizes a number of inflammatory mediators in increased amounts. These include the potent chemoattractant leukotricne B4, which seems to be responsible for the increase in chemotactic activity found in IBO mucosa, and the cysteinyl leukotrienes, which promote plasma leakage and induce edema formation. Synthesis of leukotrienes in normal and inflamed mucosa is dose-dependently inhibited by sulphasalazine, 5-aminosalicylic acid (5-ASA) and 4-aminosalicylic acid, while indomethacin, which is devoid of therapeutic efficacy in IBD patients, inhibits prostaglandin hut not leukotriene synthesis. These findings suggest that in IBD, mucosal leukotrienes may be more important inflammatory mediators than prostaglandins. ln addition to arachidonic acid-derived products, IBD mucosa generates platelet activating factor and various cytokines including interleukin-1 and tumour necrosis factor, all of which have potent proinflammatory actions. formation of most of these agents is inhibited by sulphasalazine and 5-ASA. The relative importance and the interactions of the various inflammatory mediators synthesized in IBD mucosa remain to be clarified.

<I>Salmonella enteritidis</I> SPAによる非特異的感染防御能の増強作用

The peritoneal macrophages from mice treated with soluble protective antigen (SPA) or lipopolysaccharide (LPS) showed an increase in chemotactic activity, but muramyl dipeptide (MDP)-induced macrophages did not show any enhancing effect. Conversely, the chemotactic activity of mice peritoneal neutrophils was enhanced by SPA, LPS or MDP treatment. The superoxide anion (O2-)-generating activity of SPA-induced macrophages was higher than LPS or MDP. And even 30 days after SPA treatment, a significant increase of O2(-)-generating activity was evident as compared to the control. SPA-induced macrophages showed a higher degree of intracellular killing of Listeria monocytogenes, in vitro, as compared to macrophages obtained from normal mice. In the studies of in vivo protection, the number of bacteria in the liver after challenging with L. monocytogenes was smaller in SPA-treated mice than in untreated mice. Also, SPA-treated mice showed an increased resistance to L. monocytogenes infection.

Relative contribution of leukotriene B4 to the neutrophil chemotactic activity produced by the resident human alveolar macrophage.

Human alveolar macrophages release chemotactic activity for neutrophils, providing a role for alveolar macrophages in regulating inflammation in the lung. As alveolar macrophages produce large amounts of leukotriene B4 (LTB4), a chemotactically active lipoxygenase product of arachidonic acid, we investigated the contribution of LTB4 to the total neutrophil chemotactic activity produced by these cells. Normal human alveolar macrophages were recovered by bronchoalveolar lavage from healthy volunteers and incubated either with the calcium ionophore A23187 for 1 h, or with opsonized zymosan particles or latex beads for 3 h. Nordihydroguaretic acid (NDGA), a relatively specific lipoxygenase inhibitor, blocked the release of neutrophil chemotactic activity after all three stimuli in a dose-dependent manner. This correlated with blockade of LTB4 production as measured by high performance liquid chromatography using freshly isolated alveolar macrophages, as well as blockade of [3H]LTB4 production by macrophages prelabeled with [3H]arachidonate. Molecular sieve chromatography using Sephadex G-50 confirmed that essentially all of the chemotactic activity in the stimulated macrophage supernatants co-eluted with authentic [3H]LTB4, and that NDGA completely blocked the chemotactic activity in the eluting fractions. Readdition of authentic LTB4 (1 X 10(-7) M) to the NDGA-blocked macrophage supernatants restored the chemotactic activity in the supernatants. The macrophage supernatants did not contain platelet-activating factor-like activity, as measured by the stimulation of [3H]serotonin release from rabbit platelets, and by high performance liquid chromatography. NDGA did not change the protein-secretion profiles of fresh alveolar macrophages, or of macrophages prelabeled with [35S]methionine. The complement (C) components C5adesarg were not detected in any of the supernatants by radioimmunoassay. Concentration of the supernatants by positive pressure filtration (5,000-D membrane) did not augment chemotactic activity in the stimulated supernatants or uncover chemotactic activity in the NDGA-blocked supernatants. As with the 3-h studies, when alveolar macrophages were incubated overnight with opsonized zymosan, all of the increase in chemotactic activity could also be blocked by NDGA. These data indicate that LTB4 is the predominant neutrophil chemotactic factor secreted by the normal resident human alveolar macrophage in response to two major types of stimuli, calcium fluxes across the cell membrane and the phagocytosis of opsonized particulates.

The fifth component of complement (C5) is necessary for maximal pulmonary leukocytosis in mice chronically exposed to cigarette smoke

Recruitment of elastase-containing leukocytes to the alveoli and small airways of cigarette smokers is thought to be a major contributing factor in the pathogenesis of pulmonary emphysema. The fifth component of complement (C5), when activated, acts as a potent chemoattractant for these cells. We therefore examined the effect of chronic cigarette smoke exposure on numbers of pulmonary leukocytes in congenic strains of C5-deficient (C5−) and C5-sufficient (C5+) mice. Animals were exposed daily to 15 puffs (1.5 cigarettes) or unfiltered smoke from 2A1 Kentucky Reference cigarettes. After 8 weeks of exposure, the total number of bronchoalveolar leukocytes, recovered by lavage, was significantly increased in cigarette smoke-exposed animals (both C5− and C5+) vs their sham-exposed counterparts. In addition, cigarette smoke exposure also significantly increased the total number of recovered alveolar polymorphonuclear leukocytes (PMNs) and the PMN chemotactic activity of cell-free bronchoalveolar lavage supernatants in both strains vs those of sham controls. However, total numbers of recovered bronchoalveolar leukocytes and PMNs in smoke-exposed animals were significantly greater ( P < 0.001) in C5+ mice than in their congenic C5− counterparts. In C5+ mice, acute smoke exposure caused an immediate but transient increase in chemotactic activity of lung fluids, which was not observed in C5− mice following acute smoke exposure. These results suggest that cigarette smoking induces an increase in leukocytes in the lungs of mice by mechanisms which are partly dependent on C5.

Is chemotactic activity of polymorphonuclear leukocytes increased in psoriasis?

Chemotactic activity of PMNs of 44 patients with common psoriasis and 20 healthy individuals was studied by modified Boyden chamber assay and casein as a chemoattractant. The patients were classified according to the activity of the disease, extent of skin lesions, and duration of the disease and of the last relapse. There was a high statistically significant increase in chemotactic response of the PMNs of psoriatics as compared with controls. The increase in chemotactic activity correlated positively with the activity of the disease but not with the extent of skin lesions. Very low values in some patients with longlasting and/or extensive lesions could depend upon the presence of inhibitory factors in plasma. The possible explanation for divergent results obtained by other authors could be due to the fact that various clinical parameters were not taken into account.

Neutrophil chemotaxis in psoriasis before and after PUVA therapy.

Chemokinesis and chemotaxis of neutrophil leucocytes were studied by migration under agarose in 35 patients with psoriasis and compared with 35 healthy controls. Eschericheae coli filtrate and lipopolysaccharide were used as chemo-attractants. Of these patients, 14 were tested before and after photochemotherapy. The chemotactic capacities of psoriatic serum before and after PUVA therapy were also investigated. The increase in the chemotactic activity of psoriatic polymorphonuclear leucocytes (PMNs) was not significant and remained unchanged after PUVA therapy. The activated psoriatic serum showed a slight increase in chemotactic activity when compared with normal activated serum. These results do not support an intrinsic abnormality of psoriatic PMNs, and their migration into psoriatic lesions could be chiefly due to the presence of chemotactic factors in involved epidermis.

Superoxide-dependent chemotactic activity for PMNs derived from opsonized zymosan-stimulated human platelets.

Previous studies have shown that platelets exhibit a H2O2 producing, NADH-dependent system that is activated by interaction with particulate material. Current evidence suggests that this system could be critically involved in th generation of chemotactic factor(s). In the present studies, chemotactic activity for polymorphonuclear leukocytes of supernatants derived from zymosan-stimulated human platelets has been evaluated using an agarose gel technique. Supernatants of opsonized zymosan-stimulated platelets showed significant chemotactic activity (migration index = 300 +/- 50), in comparison with supernatants prepared from platelet suspensions stimulated with nonopsonized-zymosan (migration index = 10 +/- 15) or resting platelet supernatants (migration index = 15 +/- 15). Furthermore, a marked increase in chemotactic activity of the opsonized zymosan-treated platelet supernatants was demonstrated after the addition of NADH (migration index = 525 +/- 100). The inclusion of specific inhibitors of the cycloxygenase and lipoxygenase pathways resulted in a marked reduction of chemotactic activity, which was restored in the presence of NADH. Further, the addition of superoxide dismutase completely abolished the chemotactic response induced by NADH. These data suggest that platelets are the source of chemotactic factor(s) derived from the activation of a superoxide generating system.

The chemotactic activity of native and denatured serum albumin.

Native human serum albumin (HSA) is not chemotactic for neutrophil leucocytes. Certain treatments such as acidification or reduction-alkylation may cause HSA preparations to become moderately chemotactic, whereas, after other treatments such as heat-denaturation, very little increase in chemotactic activity is observed. The chemotactic activity which follows denaturation is generally confined to monomeric forms of HSA. Acquisition of such activity correlates well with physical measurements of conformational change in the HSA molecule inasmuch as the viscosity and surface activity of chemotactically active HSA preparations are greater than those of preparations without activity. A hypothesis is proposed whereby chemotactically active sites on protein molecules are associated with hydrophobic areas of the molecule and are therefore concealed in the native state, but may become exposed during unfolding so that denatured proteins acquire chemotactic activity.