Synthetic leukotriene B4 is a potent chemotaxin but a weak secretagogue for human PMN

We have examined the effects of very pure (greater than 99.8%) chemically synthesized leukotriene B4 of verified structure on the chemotactic and secretory behavior of human polymorphonuclear leukocytes (PMN). The synthetic material is highly chemotactic and shows the same concentration dependence of this activity as does natural LTB4. Synthetic LTB4 is also a weak degranulating agent in cytochalasin B treated PMN. Maximally it released 11%, 17% and 26% as much N-acetyl-beta-D-glucosaminidase, myeloperoxidase and lysozyme as did N-formyl-methionine-leucine-phenylalanine (fMLP). Thus LTB4 differs significantly from other chemotaxins, such as C5a and fMLP, in that it is a poor secretagogue for enzymes of the specific and azurophilic granules of human PMN.     

Development of a new method of analysing chemotactic deactivation of human neutrophil granulocytes

Measurement of chemotactic migration of human neutrophil granulocytes (PMN) induced by chemotaxins serves as a simple and reliable method for assessing the expression of chemotaxin receptors. Incubation of PMN with a certain chemotaxin leads to a diminished chemotactic migration towards this chemotaxin. This is called chemotactic deactivation. We developed a new deactivation chamber to determine chemotaxis and chemotactic deactivation of human PMN. This novel chamber is a modification of the commercially available acrylic 48-well microchemotaxis chamber consisting of an upper block with wells drilled all the way through the block and a blind-well lower block. Both blocks are separated by a polycarbonate membrane. PMN from the wells in the upper block migrate through the pores of the membrane into the wells of the lower block containing the chemoattractants. Migrated PMN on the lower side of the PC membrane were quantified after staining by measuring specific light absorbance. The chemotactic activity is quantified as a ratio of stimulated migration and random migration (chemotactic index=CI). For our novel chamber, only the upper blocks of this commercial chamber were connected like a sandwich, including a polyvinylpyrrolidone-free polycarbonate membrane with a pore size of 3 microm. The wells in the upper compartment were filled with 5 x 10(4) PMN and deactivating chemotaxin. The lower block was then filled with the chemotactic stimulus and the chamber was then incubated in humidified air with 5% CO2 atmosphere at 37 degrees C. The influence of cell concentration, incubation time, chemotactic factor concentration, pore size and alkaline treatment of polycarbonate membranes on migrational activity of PMN have been investigated. The technique was rigorously standardized in order to optimize the assay conditions. The method is relatively simple, sensitive and fast. The determination of chemotaxis and deactivation are performed in the same chamber, thus avoiding cell loss due to nonspecific adherence in other incubation tubes. The chamber can be used to characterize the chemotactic activity of chemoattractants of unknown structure via known and unknown receptors. This new chamber can be very helpful in detecting unknown chemotactic stimuli, which are not detectable by, for example, antibodies.     

Leukocyte chemoattractant peptides from the serpin heparin cofactor II

Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits the coagulant proteinase alpha-thrombin. We have recently demonstrated that proteolysis of HC by catalytic amounts of polymorphonuclear leukocyte proteinases (elastase or cathepsin G) generates leukocyte chemotaxins (Hoffman, M., Pratt, C. W., Brown, R. L., and Church, F. C. (1989) Blood 73, 1682-1685). One of four peptides produced when HC is degraded by neutrophil elastase has chemotactic activity for both monocytes and neutrophils with maximal migration comparable to formyl-Met-Leu-Phe, the "gold standard" bacterially derived chemotaxin. The amino-terminal sequence of this HC peptide is Asp-Phe-His-Lys-Glu-Asn-Thr-Val-... and the peptide corresponds to Asp-39 to Ile-66 of HC. A variety of synthetic peptides derived from this sequence were evaluated for leukocyte migration activity, and a dodecapeptide from Asp-49 to Tyr-60 (Asp-Trp-Ile-Pro-Glu-Gly-Glu-Glu-Asp-Asp-Asp-Tyr) was identified as the active site for leukocyte chemotactic action. The 12-mer synthetic peptide possesses significant neutrophil chemotactic action at 1 nM (60% of the maximal activity of formyl-Met-Leu-Phe), while a peptide with the reverse sequence has essentially no chemotactic activity. Cross-desensitization experiments also show that pretreatment of neutrophils with a 19-mer peptide (Asn-48 to Ile-66) greatly reduces subsequent chemotaxis to HC-neutrophil elastase proteolysis reaction products. When injected intraperitoneally in mice, the HC-neutrophil elastase digest elicits neutrophil migration. Our results demonstrate that not only does HC function as a thrombin inhibitor, but that limited proteolysis of HC near the amino terminus yields biologically active peptide(s) which might participate in inflammation and in wound healing and tissue repair processes.     

Emigration and accumulation of PMN-leukocytes induced by endotoxin, interleukin 1 and other chemotactic substances

This publication describes polymorphonuclear leukocyte (PMN) emigration and accumulation, which is prerequisite for their defensive function in infected tissues. The extravasated PMNs can kill microorganisms, but in this process they also release proteolytic enzymes and other cell constituents which can alter and even injure the tissues, primarily the microcirculation. In the first part of the paper in vivo quantitation of the acute inflammatory reaction is described with emphasis on PMN emigration and accumulation. With 51Cr-labeled PMNs the kinetics of their emigration induced by a number of chemotaxins and chemotaxinigens was found to be similar, peaking in 1-4 hour old lesions and returning to baseline values thereafter. The most potent substance tested was endotoxin, which induced a PMN influx at a molar concentration a least 3 orders of magnitude lower than the other substances tested, implying the these substances are not the primary endogenous mediators of endotoxin induced inflammation. Next we describe an observation which shed considerable light on the mechanisms underlying PMN emigration. When a chemotaxin or endotoxin was injected intradermally and after varying periods of time reinjected into the same site, the PMN influx into those sites was diminished, compared to sites not previously injected, i. e. injected for the first time. This tachyphylaxis or diminished responsiveness was attributed to a downregulation of receptors, presumably on endothelial cells, coupled to a facilitatory mechanism. Other mechanism proposed to terminate emigration of PMNs during inflammatory reaction were unlikely, based on our experimental findings. Endotoxin is not chemotactic in vitro but it induces PMN emigration when injected intradermally. Hence the third part of the publication deals with PMN emigration induced by interleukin 1 and its significance for endotoxin-induced inflammation. IL 1 is the only chemotaxin which induces PMN accumulation at a concentration comparable to that of endotoxin and considerably lower than the other chemotaxins. There was cross tachyphylaxis between endotoxin and IL 1 and vice versa. The PMN influx into IL 1 sites injected 6 hours earlier with IL 1 or with endotoxin was diminished compared to IL 1 sites injected into normal skin. Sites injected first with IL 1 and then with a low dose of endotoxin also exhibited cross tachyphylaxis. FMLP or LTB4 injected into sites pretreated with endotoxin did not exhibit cross tachyphylaxis, i. e. the PMN influx was similar to sites injected for the first time with these chemotaxins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recombinant human tumor necrosis factor alpha lacks chemotactic activity for human peripheral blood neutrophils and monocytes

Preparations of recombinant human tumor necrosis factor alpha (rhuTNF alpha) free of aminoterminal methionine were tested for human neutrophil granulocyte (PMN) and monocyte (MO) chemotactic activity using the Boyden chamber system. Over a wide range of concentrations (10(-7)-10(-15) M) rhuTNF alpha of two different sources failed to elicit chemotactic responses in PMN or MO, whereas strong PMN and MO chemotactic activity could be detected using the tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). In addition, rhuTNF alpha containing 62% aminoterminal methionine failed to induce PMN and MO chemotaxis. It is concluded that rhuTNF alpha may not be a chemotaxin for human PMN and MO in vitro.     

Selective localization of recognition complexes for leukotriene B4 and formyl-Met-Leu-Phe within lipid raft microdomains of human polymorphonuclear neutrophils

Neutrophilic polymorphonuclear leukocytes contain glycosphingolipid- and cholesterol-enriched lipid raft microdomains within the plasma membrane. Although there is evidence that lipid rafts function as signaling platforms for CXCR chemokine receptors, their role in recognition systems for other chemotaxins such as leukotriene B4 (LTB4) and fMLP is unknown. To address this question, human neutrophils were extracted with 1% Brij-58 and fractionated on sucrose gradients. B leukotriene receptor-1 (BLT-1), the primary LTB4 receptor, partitioned to low density fractions, co-isolating with the lipid raft marker, flotillin-1. By contrast, formyl peptide receptor (FPR), the primary fMLP receptor, partitioned to high density fractions, co-isolating with a non-raft marker, Cdc42. This pattern was preserved after the cells were stimulated with LTB4 or fMLP. Fluorescence resonance energy transfer (FRET) was performed to confirm the proximity of BLT-1 and FPR with these markers. FRET was detected between BLT1 and flotillin-1 but not Cdc42, whereas FRET was detected between FPR and Cdc42, but not flotillin-1. Pretreating neutrophils with methyl-beta-cyclodextrin, a lipid raft-disrupting agent, suppressed intracellular Ca(2+) mobilization and ERK1/2 phosphorylation in response to LTB4 but had no effect on either of these responses to fMLP. We conclude that BLT-1 is physically located within lipid raft microdomains of human neutrophils and that disrupting lipid raft integrity suppresses LTB4-induced activation. By contrast, FPR is not associated with lipid rafts, and fMLP-induced signaling does not require lipid raft integrity. These findings highlight the complexity of chemotaxin signaling pathways and offer one mechanism by which neutrophils may spatially organize chemotaxin signaling within the plasma membrane.     

Secretion of novel and homologous neutrophil-activating peptides by LPS-stimulated human endothelial cells

Human umbilical vein endothelial cells in culture produce two chemotactic polypeptides when stimulated with LPS. The chemotactic factors could be purified to apparent homogeneity by HPLC techniques and were identified as 7.5-kDa and 15-kDa polypeptides by SDS-PAGE under nonreducing conditions. Both factors are potent chemotaxins for human neutrophils demonstrating half-maximal chemotaxis at 2 ng/ml and g ng/ml, respectively. In addition both peptides elicited release of azurophilic granule constituents when neutrophils were pretreated with cytochalasin B. Cross-desensitization experiments by using human neutrophils revealed cross-reactivities between both chemotaxins, not, however, with C5a or FMLP, indicating that both endothelial cell-derived neutrophil activating peptides (ENAP) are homologous. In addition, the 7.5-kDa factor (beta-ENAP) proved to be the quantitatively dominating and more potent chemotaxin as compared to the 15 kDa factor (alpha-ENAP). beta-ENAP shows biochemical and biologic similarities to monocyte- and lymphocyte-derived neutrophil-activating peptides MONAP and LYNAP, which recently were purified and sequenced.     

Potentiation of PGE1-induced increase in cyclic AMP by chemotactic peptide and Ca2+ ionophore through calmodulin-dependent processes

The interaction between prostaglandin E1 (PGE1) and chemotactic peptide formylmethionyl-leucyl-phenylalanine (fMLP) in cAMP production in guinea pig neutrophils was investigated. Both PGE1 and fMLP increased the cAMP content in neutrophils. At low concentrations of PGE1 (less than 10 nM), the effects of fMLP and PGE1 in stimulating cAMP accumulation were additive, but at high concentrations of PGE1, their effects were synergistic. The effects of PGE1 and Ca2+ ionophore A23187 instead of fMLP on cAMP accumulation were also synergistic. The synergy did not appear to be related to change in cyclic nucleotide phosphodiesterase activity, because it was still marked in the presence of isobutyl-3-methyl-1-xanthine, a phosphodiesterase inhibitor. Studies on the time course of PGE1-induced cAMP accumulation showed that cAMP production ceased within 5 min after the addition of high concentrations of PGE1. The period of cAMP production could not be prolonged by combined treatment with PGE1 and fMLP or Ca2+ ionophore A23187. The synergy was found to be caused through Ca2+-dependent processes, because depletion of the medium of Ca2+ and addition of the Ca2+ antagonist TMB-8 inhibited the synergistic increase in cAMP. Moreover, the calmodulin antagonist W-7 also effectively inhibited the synergistic increase in cAMP. These results suggest that the potentiation of PGE1-induced cAMP production by fMLP or Ca2+ ionophore A23187 is catalyzed by calmodulin-dependent processes. However, the synergistic increase in cAMP production was not inhibited by arachidonic acid cascade inhibitors such as indomethacin, BW755C, or nordihydroguiaretic acid, and a combination of PGE1 and a protein kinase C activator, tetradecanoyl phorbol acetate (TPA), did not cause synergistic increase in cAMP. Marked increase in cAMP was also induced by a combination of cholera toxin and fMLP or Ca2+ ionophore A23187, but not by a combination of forskolin and fMLP or Ca2+ ionophore A23187. The synergistic increase in cAMP was not sustained in isolated membranes. On the contrary, PGE1-induced cAMP production in isolated membranes was suppressed by their pretreatment with fMLP or Ca2+ ionophore A23187. These data suggest that the synergistic effects of PGE1 and fMLP or Ca2+ ionophore in increasing the cAMP level are due to potentiation of PGE1-induced cAMP production by Ca2+ and calmodulin-dependent processes.     

Monocyte Chemoattractant Activity of Ser → Ala Active Site Mutant Recombinant α-Thrombin

α-Thrombin is chemotactic for human monocytes with optimal activity between 10-100 nM. The mechanism by which this response is mediated remains a point of controversy. The purpose of this study was to compare the chemotactic activity of proteolytically inactive thrombin (active site Ser¹⁹⁵ → Ala mutant or Phe-Pro-Arg-chloromethyl ketone-inactivated thrombin) to thrombin and the "tethered ligand" thrombin receptor agonist peptide SFLLRN (single-letter amino acid code). Monocyte chemotaxis was compared to an optimal concentration (10 nM, considered to be 100%) of formyl-Met-Leu-Phe (fMLP). Proteolytically inactive thrombin (38% of fMLP) had similar chemotactic activity to active thrombin (46% of fMLP) at a concentration of 100 nM. Chemotaxis to SFLLRN was comparable to that of a control hexapeptide (FSLNLR) which is not an agonist for the tethered ligand thrombin receptor. Cross-desensitization experiments showed that pretreatment of monocytes with either mutant or active thrombin reduced subsequent chemotaxis to both thrombin chemotaxins. Pretreatment with SFLLRN did not decrease subsequent chemotaxis to either form of thrombin. Calcium flux measurements showed that both active thrombin and SFLLRN induced a rapid increase in monocyte and platelet intracellular calcium concentration. However, there was no intracellular calcium change in response to mutant thrombin or FSLNLR. Likewise, active thrombin and SFLLRN induced a rapid net increase in polymerized actin, but mutant thrombin and FSLNLR did not. By contrast, both active and mutant thrombin induced a polarization of monotocyte morphology and actin distribution. This polarization has been associated with directed migration in many cell types. SFLLRN, however, induced a symmetrical increase in polymerized actin. These results suggest that measurements of intracellular calcium and polymerized actin are not perfect surrogate tests for true chemotactic activity. These results show that thrombin proteolysis is not required for monocyte chemotaxis and may be mediated by interaction with a binding site other than the tethered ligand thrombin receptor.     

Polymorphonuclear leukocytes modulate tissue factor production by mononuclear cells: role of reactive oxygen species

To determine whether polymorphonuclear leukocytes (PMN) modulate the production of tissue factor (TF) by monocytes, PBMC were incubated with increasing concentrations of PMN. PMN did not express any procoagulant activity. After 20-h cocultures, PMN enhanced or inhibited the TF production of PBMC, and this effect depended on the PMN/PBMC ratio. When the ratio increased from 1/1000 to 1/5, without or with LPS, the TF activity of PBMC increased to peak at 2.5-fold the baseline value (p < 0.01). The TF Ag and TF mRNA also increased. This potentiating effect was mediated by reactive oxygen species (ROS) released by PMN during the coculture; it did not require direct cell contact between PMN and PBMC, it was enhanced when PMN were stimulated by fMLP (a chemotactic peptide), and it was inhibited by two antioxidants, N-acetyl cysteine and pyrrolidine dithiocarbamate. In contrast, when the PMN/PBMC ratio was further increased from 1/2 to 2/1, the PBMC TF activity, Ag, and mRNA decreased and were inhibited compared with those of PBMC cultured alone (p < 0.01). This inhibitory effect required direct cell contact between PMN and PBMC, and it was not due to a PMN-mediated cytotoxicity. To confirm the role of ROS, H2O2 enhanced then inhibited the TF activity of PBMC in a dose-dependent manner, similarly to PMN. Thus, PMN may play an important role in the pathogenesis of thrombosis and atherosclerosis by exerting concentration-dependent regulatory effects on the TF production by PBMC via the release of ROS.     

cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.     

Response of cyclic AMP by DMSO differentiated HL-60 cells exposed to electric interferential current after prestimulation

The action of interferential current (IFC) upon intracellular content of cyclic adenosine monophosphate (cAMP) after prestimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) has been investigated. Human promyelocytes (HL-60) differentiated to granulocytes by dimethylsulphoxide (DMSO) have been treated with different concentrations of fMLP. This enhances their cAMP content. The half maximal effective concentration (EC50) was about 4 nM. Exposure to IFC with modulation frequencies of 35 and 125 Hz (5 min, 250 microA/cm2) after prestimulation with various concentrations of fMLP had no enhancing effect at low or high concentrations of fMLP. In contrast, at medium concentrations in the range of about 100 pM fMLP, a significant enhancement of cAMP could be observed. This synergistic effect of fMLP and IFC has been examined in detail by varying the modulation frequency, current density, and exposure time. Enhancement of cAMP content could be observed at certain modulation frequencies and exposure times suggesting a window effect, whereas for the current density in the range measured (8.5 microA/cm2-2.5 mA/cm2) a constant enhancement could be observed. The synergistic effect of fMLP and IFC could only be observed in the treatment sequence of fMLP followed by IFC; an inverse sequence had no effect on the cAMP content. .     

Effects of colchicine, vinblastine and nocodazole on polarity, motility, chemotaxis and cAMP levels of human polymorphonuclear leukocytes

We present evidence for intrinsic polymorphonuclear leukocyte (PMN) polarity manifested in presence of microtubule-disrupting drugs. Polarization in response to colchicine correlated with the known dose-dependent effects of this drug on microtubule disassembly. The response to 10(-5) M colchicine, 10(-5) M vinblastine and 10(-6) M nocodazole was associated with stimulated motility and random locomotion. Responses elicited by microtubule-disrupting drugs differed from f-Met-Leu-Phe (fMLP)-induced polarization by functional and morphological criteria. Polarization, motility and orthokinesis responses were much weaker. Furthermore, ruffling was almost absent in PMNs polarized in response to colchicine, vinblastine or nocodazole. The response was inhibited by cytochalasin B, indicating that it is microfilament-dependent. We suggest that microtubule-disrupting drugs induce motility via structural changes in the cytoskeleton which act as signals for the motor apparatus. The intrinsic polarity manifested in the presence of microtubule-disrupting drugs could be reversed by an extracellular chemotactic gradient. Stimulated locomotion and motility in response to microtubule-disrupting drugs was only observed with initially spherical PMNs but not with initially motile cells. The findings provide an explanation for the numerous conflicting statements on the chemokinetic activities of these drugs. The role of cAMP in stimulated polarization and motility has been studied. Colchicine, vinblastine and nocodazole elicited a transient elevation of cAMP levels within 1 min of stimulation. cAMP elevation and stimulated motility were not quantitatively correlated.     

Receptor-operated activation of polymorphonuclear leukocytes: different effects of NAP-1/IL-8 and fMet-Leu-Phe or C5a.

We examined the production of PAF and LTB4 by PMN in response to NAP1/IL-8 alone, or after preincubation with GM-CSF, which has been shown to enhance PMN responsiveness and to prime PMN for production of those bioactive lipids. NAP-1/IL-8 does not induce the synthesis of PAF and LTB4 from endogenous phospholipid precursors, even after preincubation with GM-CSF. In addition and again in contrast to fMLP and C5a, NAP-1/IL-8 fails to induce an enhanced oxidative burst in GM-CSF primed PMN. Exogenously added PAF or LTB4 can mimic the priming effect of GM-CSF for an enhanced oxidative burst in response to all examined chemotactic peptides including NAP1/IL-8. Our data reveal a possible autocrine role of PAF and LTB4 in the enhanced responsiveness of GM-CSF primed PMN towards fMLP or C5a, but not NAP-1/IL-8.     

Deactivation of guinea pig pulmonary alveolar macrophage responses to N-formyl-methionyl-leucyl-phenylalanine: chemotaxis, superoxide generation, and binding

Incubation of pulmonary alveolar macrophages (PAM) with the synthetic chemotactic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) results in deactivation of PAM chemotaxis. The chemotactic response to 10(-8) M FMLP was inhibited 85% after 30 min of preincubation with 10(-6) M FMLP and 48% by 10(-8) M FMLP. Only the higher dose of FMLP (10(-6) M) caused deactivation of the chemotactic response to C5a (20%). Preincubation with partially purified C5a at a concentration of 100 microliter/ml produced a 32% inhibition of the PAM response to 10(-8) M FMLP. In contrast, preincubation with FMLP had no significant effect on superoxide generation, either at baseline or after stimulation. Levels of intracellular cyclic adenosine-3',5'-monophosphate (cAMP) increased in response to PGE1 in the presence of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, but FMLP failed to induce a change in cAMP levels. Studies of 3H-FMLP binding were consistent with two populations of membrane receptors with different affinities. Preincubation of PAM with FMLP did not result in a reduction of maximal binding. We conclude that FMLP induces deactivation of PAM chemotaxis, but cross-deactivation occurs only after high dose treatment. Unlike the PMN, macrophage chemotactic activation is not accompanied by an elevation in cAMP levels. These observations suggest that PAM chemotaxis is influenced by prior exposure to chemotactic stimuli, but other aspects of the PAM response diverge from that of PMN. The mechanism of deactivation of PAM does not appear to result from a shift in the dose-response curve or decreased availability of membrane receptors, but may involve uncoupling of post-receptor cellular responses.     

An indirect bioluminescence method for the quantitative measurement of polymorphonuclear cell chemotaxis

A method is presented which allows the quantification of the effects of chemotactic factors on polymorphonuclear leukocytes on the basis of a sensitive ATP measurement using bioluminescence. The assay measures those cells which have migrated through a commercial 3 μm filter system (Transwell?). The assay was tested under standardized conditions with different chemotactic agents (leukotriene B4 [LTB4], N-formyl-methionyl-leucyl-phenylalanine [FMLP], N-formyl-methionyl-leucyl-phenylalanine-methyl ester [M-FMLP]). Under appropriate conditions the migration of PMN-cells is time-dependent and linear for 60 minutes. Spontaneous migration of PMN cells is simultaneously quantified in a simple way, and the value obtained allows a determination of the actual chemotactic stiuation of the PMN cells. In healthy humans the spontaneous migration varied between 4.2% and 14.4% of the total number of PMN cells. An optimal chemotactic activity was detected at 10^?8/mol/I for FMLP and 10^?7 mol/l for M-FMLP in PMN leukocytes, which correlates with literature values. It was also found that in contrast to EDTA blood, heparinized blood lowers the ATP level of PMN cells (by about 50%) and therefore heparinized blood is not recommended for chemotactic experiments. This assay is a simple tool for quantification of the spontaneous migration, and the chemotactic response to specific factors and their inhibitors in particular for pharmacological experiments. In contrast to the ‘classical’ chemotactic assays this method also permits the simultaneous testing of the influence of chemotactic substances on cellular ATP levels.     

Thioredoxin specifically cross-desensitizes monocytes to MCP-1

Thioredoxin (Trx) is a protein disulfide oxidoreductase which can be secreted and acts as a cytokine. As we recently reported that Trx is chemotactic, we investigated whether it desensitizes monocytes or PMN to other chemokines. Preincubation for 15 min with Trx inhibited the chemotactic response of monocytes to MCP-1, but not to fMLP. This effect was independent of whether Trx was present during the chemotaxis assay or only during the preincubation. Preincubation (5 min) with Trx also inhibited the increase in intracellular Ca(2+) induced by MCP-1 in monocytes, but not that induced by fMLP. Preincubation with Trx did not affect the chemotactic response induced in PMN by IL-8. The inhibition of chemotactic and Ca(2+) responses to MCP-1 in monocytes was not due to a down-regulation of the MCP-1 receptor, as shown by receptor binding studies. The Ca(2+) response to MCP-1 was also inhibited by Trx in a CCR2-transfected cell line. It is suggested that Trx inhibits monocyte responses to chemokines by acting downstream of the chemokine receptors. Since there are high concentrations of circulating Trx in infection and inflammatory diseases, this might act as an inhibitor of monocyte migration in vivo.     

Synthetic leukotriene B4 is a potent chemotaxin but a weak secretagogue from human PMN

We have examined the effects of very pure (> 99.8%) chemically synthesized leukotriene B₄ of verifeid structuer on the chemotactc and secretry behavior of human polymphonuclear leukocytes (PMN). The synthetic material is highly chemotactic and shows the same concentration dependence of this activity as does natural LTB₄. Synthetic LTB₄ is also a weak degranulating agnet in cytochalasin B treated PMN. Maximally it released 11%, 17% and 26% as much N-acetyl-β-D-dlucosaminidise, myeloperoxidase and lysozyme as did N-formyl-methionine-leucine-phneylalanine (fMLP). Thus LTB₄ differs significantly from other chemotaxisn, as such as C5a and fMLP, in that it is a poor secretagogue for enzymes of the specific adn azurophilic granules of human PMN.     

Purification and partial biologic characterization of a human lymphocyte-derived peptide with potent neutrophil-stimulating activity

A novel neutrophil-activating peptide is detected in supernatants from mitogen-stimulated human T lymphocyte preparations. This chemotaxin was purified to apparent homogeneity by sequential Gel G-75 permeation chromatography, wide pore reversed phase (RP-8) HPLC, size exclusion HPLC, and reversed phase (RP-18) HPLC. Additional characterization of this lymphocyte-derived neutrophil-activating peptide (LYNAP) resulted in a single peak upon reversed phase HPLC and size exclusion HPLC. SDS-PAGE under nonreducing conditions revealed a single line at 10 kDa. LYNAP stimulated neutrophil chemotaxis (ED50 of 3 +/- 3 ng/ml), chemokinesis (ED50 of 2 +/- 2 ng/ml), and caused degranulation of cytochalasin B pretreated human neutrophils (ED50 of 20 ng/ml). In purified human monocytes, chemotactic responses to LYNAP at doses up to 100 ng/ml were absent, indicating nonidentity with a lymphocyte-derived monocyte chemotactic factor previously described by other workers. LYNAP shows biochemical and biologic similarities to a recently detected monocyte-derived neutrophil-activating peptide (MONAP). Moreover, desensitization experiments revealed cross-deactivation between LYNAP and MONAP, not, however, between these two chemotactic peptides and other well characterized polymorphonuclear leukocyte chemotaxins, e.g., C5a, FMLP, leukotriene B4, or platelet-activating factor. This finding points toward structure identity or homology of both chemotaxins, MONAP and LYNAP.     

The TNF Receptors p55 and p75 Mediate Chemotaxis of PMN Induced by TNFalpha and a TNFalpha 36-62 Peptide

The present study was performed to examine whether residues 36-62 of TNFalpha contain the chemotactic domain of TNFalpha, and whether the p55 and p75 TNF receptors are involved in TNFalpha induced chemotaxis. The chemotactic effect of TNFalpha on PMN was inhibited by the mAbs Hrt-7b and Utr-1, against the p55 and p75 TNF receptors, respectively. Both receptors may therefore be required for mediating the chemotactic effect of TNFcz. The synthetic TNFalpha 36-62, similar to TNFalpha, had chemotactic effects on both PMN and monocytes. The chemotactic activity of the TNFalpha 36-62 peptide on PMN, was inhibited by Htr-7b, Utr-1 and soluble p55 receptor, which shows that the peptide possessed the ability to induce chemotaxis through the TNF receptors. In contrast to TNFalpha, the peptide did not show a cytotoxic activity against WEHI 164 flbrosarcoma cells. It is suggested that different domains of the TNFalpha molecule induce distinct biological effects.