A cathelicidin family of human antibacterial peptide LL-37 induces mast cell chemotaxis

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 in the local milieu occurs, and such accumulation requires directed migration of this cell population. As it has previously been reported that the human cathelicidin-derived antibacterial peptide, LL-37, stimulates the degranulation of mast cells, we hypothesized that LL-37 could be a mast cell chemotaxin. The present study shows that LL-37 is a potent chemotactic factor for mast cells. The chemotactic response was dose-dependent and bell-shaped, reaching an optimal concentration of 5 microg/ml. In addition, checkerboard analysis showed that cell migration towards this peptide was chemotactic rather than chemokinetic. Moreover, Scatchard analysis using 125I-labelled LL-37-derived peptide revealed that LL-37 has at least two classes of receptors, namely high- and low-affinity receptors, on mast cells. Furthermore, the competitive binding assay suggested that LL-37 is unlikely to utilize formyl peptide receptor-like 1 (FPRL1), a functional LL-37 receptor for neutrophil and monocyte migration, on mast cells. In addition, the treatment of cells with pertussis toxin and phospholipase C inhibitor, U-73122, inhibited LL-37-mediated migration, indicating that LL-37 induces mast cell chemotaxis through a Gi protein-phospholipase C signalling pathway. These results show that besides its antibacterial activities, LL-37 may have the potential to recruit mast cells to inflammation foci.     

Differential expression of binding sites for chemokine RANTES on human T lymphocytes

The C-C chemokine RANTES, a T lymphocyte chemoattractant, is considered an important mediator of inflammation, allergy, and host defense against HIV-1 infection. In this study, we investigated the modulation of binding of RANTES to T lymphocytes. Human peripheral blood CD3+ T cells, when freshly isolated from buffy-coat blood, expressed a considerable number of high-affinity binding sites for RANTES. These cells also showed significant chemotactic migration in response to RANTES in vitro. After 6–15 h incubation at 37°C, the binding of RANTES, but not of macrophage inflammatory protein-1α (MIP-1α) or of monocyte chemotactic protein-3 (MCP-3), consistently increased. Scatchard analyses indicated that the number of binding sites for RANTES increased about threefold by 15 h without any change in the affinity. The increase in RANTES binding was no longer detected by 24 h. This increase in the specific binding was mainly attributable to CD4⁺ T cells and was not associated with increased chemotactic activity of these cells in response to RANTES. Incubation with anti-CD3 antibody for 15 h markedly reduced the binding capability of T cells for RANTES and was associated with decreased chemotactic activity. On the other hand, when T cells were incubated with interleukin-2 (IL-2) for 1 week, the specific binding for all three C-C chemokines, RANTES, MIP-1α, and MCP-3 was markedly increased in comparison to cells cultured in the absence of IL-2. These results suggest that the expression of binding sites on T cells for RANTES is differentially modulated, indicating the existence of novel receptors for RANTES that do not bind MIP-1α.     

Chemotactic activity of C5ades Arg: Evidence of a requirement for an anionic peptide ‘helper factor’ and inhibition by a cationic protein in serum from patients with systemic lupus erythematosus

Sera from some patients with systemic lupus erythematosus (SLE)¹ contain a uniquely specific, reversible inhibitor of complement (C5)-derived chemotactic activity for polymorphonuclear leukocytes. SLE serum, proven capable of significantly inhibiting C5-derived chemotactic activity in zymosan-treated serum (ZTS), was found incapable of inhibiting the chemotactic activity of the highly purified human anaphylatoxin, C5a (10–20 ng/ml). Similarly, SLE serum was found incapable of inhibiting the chemotactic activity generated in ZTS containing the carboxypeptidase inhibitor, epsilon amino-caproic acid (EACA). EACA protects C5a from the action of the anaphylatoxin inactivator in serum and thereby prevents conversion of C5a to a peptide (C5a_{des Arg}) which is completely devoid of anaphylatoxin activity. Highly purified human C5a_{des Arg} (40–160 μg/ml) was not chemotactic unless assayed in the presence of small amounts of normal human serum. The ‘helper factor’ in normal human serum which permits C5a_{des Arg} to exhibit chemotactic activity was isolated and found to be an anionic polypeptide. The chemotactic activity of C5a_{des Arg} plus normal serum was inhibited significantly by SLE serum. The inhibitor in SLE serum was isolated and determined to be a 69,000 mol. wt cationic protein. These data suggest strongly that the cationic inhibitor in SLE serum acts not on C5a but only on the ‘complex’ of C5a_{des Arg} plus a specific peptide ‘helper factor’. This ‘complex’ accounts for a substantial proportion of the chemotactic activity in ZTS.     

Calcitonin gene-related peptide (CGRP) activates human neutrophils--inhibition by chemotactic peptide antagonist BOC-MLP.

The effect of the neuropeptides substance P, neurokinin A and alpha-calcitonin gene-related peptide (CGRP) on human neutrophil granulocytes was investigated. Substance P induced secondary granule secretion at a concentration of 100 microM. CGRP induced a significant secretory response at 10 microM and thus appeared to be about 10 times more potent than substance P. Calcitonin and a fragment of CGRP, CGRP(8-37), had no effect on neutrophil degranulation. The chemotactic peptide antagonist BOC-MLP (100 microM) inhibited lactoferrin secretion mediated both by CGRP and chemotactic peptide FMLP almost completely, while secretion in response to tumour necrosis factor (TNF) was unaffected. Results from receptor binding studies showed that CGRP and N-formyl-methionyl-leucyl-phenylalanine (FMLP) do not compete for binding. This indicates that CGRP does not exert its effects by binding to the chemotactic peptide receptor. CGRP induced a rapid increase in the cytosolic-free calcium concentration and this increase was not, unlike that induced by FMLP, abolished by preincubation of the cells with pertussis toxin (1000 ng/ml). Therefore CGRP signal transduction in neutrophils appears to involve rapid changes in the cytosolic-free calcium concentration but not a pertussis toxin-sensitive G-protein. In summary, this is the first report to show that CGRP can directly activate neutrophil granulocytes, and this probably occurs via a cell surface receptor which is distinct from that of FMLP although both the CGRP and FMLP-mediated effects can be blocked by BOC-MLP.     

Synthetic peptide chemotactic factors for neutrophils: the range of active peptides, their efficacy and inhibitory activity, and susceptibility of the cellular response to enzymes and bacterial toxins.

The chemotactic activity for neutrophil leucocytes of twenty-six peptides of varied sequence, of which the majority were N-formylated, was assessed by determining the concentration at which each was maximally active and the efficacy of each peptide at that concentration. These two measures of activity did not correlate with one another. Many formylated peptides with a wide variety of sequences were active. Of these, the formyl-methionyl peptides had highest efficacy, but many other peptides were active at concentrations as low as the formyl-methionyl tripeptides. Unrelated peptides, viz formyl-methionyl-leucyl-phenylalanine, acetyl-tri-alanine, formyl-tri--phenyla-lanine, cross-inhibit the cells' response to one another, and this inhibition is reversible. Inhibition is prevented if the cells are incubated throughout the experiment in levamisole or A23187. These experiments suggest that the leucocyte peptide receptor is capable of binding many ligands, and that activation of a response is not solely a function of binding affinity. They exclude a strict steric specificity for binding. Chemotactic responses to formylated peptides were shown to be reduced in cells pretreated with perfringolysin, a bacterial cholesterol-binding toxin, and with phospholipase C. Trypsin and pronase also reduced these responses when used at 500 micrograms per 10(6) cells but not at lower doses.     

Chemotactic factor binding and functional capacity: a comparison between human granulocytes and differentiated HL-60 cells

In the presence of dimethyl sulfoxide (DMSO), the leukemic promyelocytic cell line HL-60 will differentiate into mature polymorphonuclear granulocytes. In the present report, we compare chemotactic factor binding and function in HL-60 cells with that of normal human granulocytes using the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) as ligand. The cellular response measured as CL was changed as a result of storage or conditioning of normal peripheral blood cells. With these cells, a conditioning procedure at room temperature resulted in a pronounced increase in the CL response. The increase of the CL response was probably a result of increased expression of cryptic receptors, since the changes of the oxidative response to FMLP was accompanied by increased binding of the peptide to the cell surface. Scatchard analysis revealed that the increased binding was due to an increased number of receptors. In differentiated HL-60 cells, conditioning neither led to increased production of oxidative metabolites, nor to any increased binding of the peptide. The data thus indicate that many FMLP receptors could reside in a cryptic site that is not accessible to extracellular ligands, and that conditioning results in an increased exposure of these receptors, followed by an increased oxidative response to the ligand in normal cells but not in mature HL-60 cells.     

Chemotaxis of mitogen-activated human lymphocytes and the effects of membrane-active enzymes.

Following incubation with polyclonal activators such as PHA or concanavalin A, human peripheral blood lymphocytes show directional migration towards chemotactic factors such as endotoxin-activated plasma, casein or denatured proteins. This migration is inhibited by treatment of the cells with phospholipase C and sphingomyelinase C, but little affected by proteases or glycosidases. In addition, these lymphocytes migrate towards PHA and other lymphocyte activators when these reagents are used as chemoattractants at concentrations well below their mitogenic doses. They also migrate towards staphylococcal protein A. Migration towards PHA and protein A is reduced by pretreating the lymphocytes with proteases but not with phospholipase C. These results suggest two independent membrane interactions which initiate lymphocyte chemotaxis, one acting directly via the lipid bilayer, the other involving the binding of ligands to membrane proteins.     

Casein-mediated neutrophil chemotaxis

This study was directed toward demonstrating an interaction between human polymorphonuclear leukocytes (PMNs) and the chemotactic factor, casein. Experiments conducted using fluorescein-labeled casein indicated that PMNs have a membrane receptor for this chemotactic factor. This receptor was specific for casein and was not found on lymphocytes. A direct parallel was shown between the binding of FITC-casein and the PMN chemotactic response to this protein. Binding to the receptor was optimal at 25°C, unaffected by sodium azide, and partially reduced by iodoacetate. Enzymatic treatment of PMNs with pronase and trypsin resulted in a loss of both FITC-casein-mediated PMN fluorescence and chemotaxis. Our data indicate that human PMNs have a membrane receptor for casein and that interaction with this receptor results in a chemotactic response.Supported in part by PHS grants HL 17179, #1 RO1 CA20819-01, AMAI13824-06, AM 18487-01, and in part by the Arthritis Foundation.     

A synthetic peptide which specifically inhibits heat-treated interleukin-8 binding and chemotaxis for neutrophils

Interleukin-8 (IL-8) is a peptide which is secreted by stimulated human monocytes and which is chemotactic for human neutrophils. We synthesized three overlapping peptides spanning the amino-terminal region of the IL-8 sequence. None of the peptides retained the chemotactic activity of the native molecule. One of the peptides, IL-8(_3–25), inhibited the neutrophil chemotactic activity of recombinant IL-8 (rIL-8) which had been preheated to 40°C but did not reduce neutrophil chemokinesis, or the chemotactic activity of unheated rIL-8, FMLP, C5a or LTB₄. Interleukin-8 exhibited similar binding kinetics and chemotaxis for neutrophils regardless of whether it had been pretreated at 40°C.In addition, IL-8(_3–25) was also able to decrease the binding of prehead IL-8 to neutrophils. IL-8(_3–25), which can self-associate, binds directly to receptors on the neutrophil. The data suggest that heat-treated, but not untreated, IL-8 causes the IL-8(_3–25) multimers to disaggregate, allowing the monomeric peptide to directly bind to the IL-8 receptor and thus inhibiting IL-8/receptor binding.     

Characterization of the rat neutrophil formyl peptide chemotaxis receptor.

Numerous synthetic N-formylated peptides, believed to be the analogs of the naturally occurring initiating signal peptides produced by bacteria, are potent chemotactic agents for phagocytic cells in several species. The authors have characterized the receptor with moderately high affinity for the chemotactic peptide f-Met-Leu-[3H]Phe on the rat peritoneal neutrophils. When neutrophils are incubated with f-Met-Leu-[3H]Phe at 24 C, the binding is saturable and reversible. The receptor on the inflammatory rat neutrophils has an equilibrium dissociation constant (KD) of 3.4 x 10(-8) M at 24 C, and there are approximately 65,000 sites per cell. In addition, the potency of several of these chemotactic peptides in inducing lysosomal enzyme secretion and superoxide production correlated well with their ability to compete with f-Met-Leu-[3H]Phe for receptor binding. Structure activity studies further demonstrate that the fine specificity of the formyl peptide receptor has been conserved across species lines.     

Modulation of C5aR expression on human neutrophils by encapsulated and acapsular Cryptococcus neoformans

Cryptococcus neoformans and cryptococcal surface polysaccharides influenced C5aR expression on human polymorphonuclear neutrophils (PMN). Encapsulated and acapsular strains produced dramatically different effects. Treatment of PMN with acapsular cryptococci up-regulated C5aR expression; whereas treatment with encapsulated cells suppressed C5aR expression. Glucuronoxylomannan (GXM), the principal constituent of the cryptococcal capsule, was responsible for such inhibition. Increased C5aR expression following treatment with acapsular cryptococci was accompanied by increased binding of C5a to PMN, increased superoxide production in response to stimulation with C5a, and an increased chemotactic response to C5a. Conversely, decreased C5aR expression following treatment with encapsulated cryptococci or acapsular cryptococci that had been pretreated with GXM was accompanied by decreased binding of C5a to PMN and a decreased chemotactic response to C5a. Our results raise the possibility that the down-regulation of C5aR expression by encapsulated cryptococci might alter PMN function at the site of cryptococcal infection.     

Chemotactic activity of collagen-like polypeptides for human peripheral blood neutrophils

Damage to interstitial connective tissue is associated with the rapid accumulation of monocytes and neutrophils at the site of injury. To study the role of collagen fragments in neutrophil migration, we analyzed the chemotactic properties of peptide fragments of bovine collagen digested with bacterial collagenase or cyanogen bromide and small molecular weight synthetic polypeptides containing proline (Pro), hydroxyproline (Hyp), and glycine (Gly), the major amino acids that comprise collagen. Using the Boyden chamber and under agarose techniques, we found that collagen fragments were as potent in inducing chemotaxis in neutrophils as the bacterial-derived peptide f-met-leu-phe. The synthetic polytripeptides (Pro-Pro-Gly)5 and (Pro-Hyp-Gly)5 were found to be equipotent in inducing chemotaxis, producing a maximal induction of chemotaxis at 5-10 nM. This suggests that Hyp, the unique imino acid found in collagen, is not required for chemotactic activity. Increasing the length of the synthetic tripeptide from 5 to 10 subunits decreased its chemotactic activity, while the single tripeptide subunit (Pro-Hyp-Gly)1 was the least active peptide, inducing a maximal response at 100 nM. To study the structural requirements for chemotaxis, Pro-Hyp-Gly tripeptides were synthesized with modifications at the N and C terminals ends. Addition of a methyl group to the carboxyl of Gly to form an ester enhanced the chemotactic activity of the peptide by 50%, while substitutions on the amino terminus with an acetyl group decreased the chemotactic activity by 50%. Substitution on the amino terminus with a Boc group decreased the chemotactic activity by 100%. These results indicate that there are specific structural requirements for chemotaxis induced by peptides having a collagen-like sequence of amino acids.     

Modulation of human eosinophil polymorphonuclear leukocyte migration and function.

Eosinophil migration toward a concentration gradient of a chemotactic factor is regulated at four levels. Diverse immunologic pathways generate stimuli with eosinophil chemotactic activity, including the complement products C5a and a fragment of C3a and the peptide products of mast cells and basophils activated by IgE-mediated reactions, such as eosinophil chemotactic factor of anaphylaxis (ECF-A) and other oligopeptides. The intrinsic preferential leukocyte activity of the chemotactic stimuli represents the second level of modulation, with ECF-A and other mast cell-derived peptides exhibiting the most selective action on eosinophils. The third level of control of eosinophil chemotaxis is composed of inactivators and inhibitors of chemotactic stimuli and is exemplified by degradation of C5a by anaphylatoxin inactivator or chemotactic factor inactivator and of ECF-A by carboxypeptidase-A or aminopeptidases. The activity of ECF-A is uniquely suppressed by equimolar quantities of its NH2- terminal tripeptide substituent, presumably by eosinophil membrane receptor competition. Factors comprising the fourth level of regulation, which alter eosinophil responsiveness to chemotactic stimuli, include the chemotactic factors themselves, through deactivation; nonchemotactic inhibitors such as the COOH-terminal tripeptide substituent of ECF-A, the neutrophil-immobilizing factor (NIF), the phagocytosis-enhancing factor Thr-Lys-Pro-Arg, and histamine at concentrations greater than 400 ng/ml; and nonchemotactic enhancing principles represented by ascorbate and by histamine at concentrations of 30 ng/ml or less. Local concentrations of eosinophils called to and immobilized at the site of a hypersenitivity reaction may express their regulatory functions by degrading the chemical mediators elaborated including histamine, slow-reacting substance of anaphylaxis (SRS-A), and platelet-activating factor (PAF) by way of their content of histaminase, arylsulfatase B, and phospholipase D, respectively. Immunologic pathways may thus provide the capability for early and specific host defense reactions with a later influx of eosinophils preventing irreversible local tissue alterations or distant organ effects.     

Association of aberrant F-actin formation with defective leukocyte chemotaxis and recurrent pyoderma

A young boy with recurrent skin infections and slow wound healing was shown to have an isolated leukocyte chemotactic defect. The chemotactic abnormality was persistent throughout the observation period, could be demonstrated both in vivo and in vitro, and was not related to known causes of chemotactic defects. To investigate the underlying pathogenetic mechanism for this abnormality, the patient's polymorphonuclear (PMN) leukocytes were studied for their ability to respond to the chemotactic peptide N-formyl-methionylleucylphenylalanine (FMLP). The patient's leukocytes were able to bind FMLP normally and responded appropriately to the stimulus as shown by a rise in intracellular calcium after binding. However, his PMN leukocytes demonstrated abnormalities in the formation and disassembly of filamentous actin (F-actin), an important structural component in cell locomotion. Since the formation and disassembly of F-actin are important in the recycling of actin and crucial in the cell movement, the observed abnormalities may account for the disorder of chemotaxis seen in this patient. The findings in this case resemble the syndrome of neutrophil actin dysfunction. However, observed differences, including a much milder clinical disease, distinguish between these two clinical entities.     

Epithelial cell-derived human beta-defensin-2 acts as a chemotaxin for mast cells through a pertussis toxin-sensitive and phospholipase C-dependent pathway

Mast cells are known to accumulate at the sites of inflammation in response to chemoattractants generated in the local milieu. Since human beta-defensin-2 (hBD-2) is generated in several epithelial tissues where mast cells are present and because we have recently reported that this human antibacterial peptide induces mast cell degranulation, we thus hypothesized that hBD-2 could be a mast cell chemotaxin. Here we report that hBD-2 directly and specifically induces mast cell migration with an optimal concentration of 3 microg/ml. Checkerboard analysis showed that the migration was more chemotactic rather than chemokinetic. Moreover, Scatchard analysis using 125I-labeled hBD-2 revealed that mast cells have at least two classes of receptors, high- and low-affinity receptors, for this peptide. Moreover, the competitive binding assay suggested that hBD-2 is unlikely to utilize CCR6, a functional receptor for hBD-2-mediated dendritic and T cell migration, on mast cells. In addition, treatment of mast cells with G protein inhibitor, pertussis toxin, and phospholipase C inhibitor, U-73122, abolished the cell chemotaxis in response to hBD-2, indicating that the G protein-phospholipase C signaling pathway is involved in hBD-2-induced mast cell activation. Thus, we suggest that hBD-2, which was originally believed to be involved in innate host defense, may participate in the recruitment of mast cells to inflammation foci.     

Requirements for the different cysteines in the chemotactic and desensitizing activity of human thioredoxin

Thioredoxin (Trx) is a protein disulfide oxidoreductase that can be secreted and act as a chemoattractant for leukocytes. Like chemokines, it causes desensitization of monocytes against its chemotactic activity and that of monocyte chemoattractant protein-1 (MCP-1). To investigate the role of the redox properties of Trx, and particularly of some of its five cysteines, in its chemotactic and desensitizing action, we tested different mutants, including Trx80, a truncated form, and various mutants lacking specific cysteines: Trx C62S/C73S and the redox-inactive mutant Trx C32S/C35S. Of the mutants, only Trx80 maintained the chemotactic activity of wild-type Trx toward both monocytes and polymorphonuclear neutrophils, all of them desensitized monocytes against wild-type Trx or MCP-1, but not chemotactic peptide formyl-methionyl-leucil peptide. These data indicate that different redox-active cysteines are important for Trx chemotactic action, whereas its desensitizing action does not have these requirements, suggesting a redox-independent mechanism.     

Characteristics of binding of a potent chemotactic formyl tetrapeptide, formylmethionyl-leucyl-phenylalanyl-phenylalanine, to the receptors on rabbit neutrophils

Binding of a potent chemotactic formyl tetrapeptide, formylmethionyl-leucyl-phenylalanyl-phenylalanine (fMet-Leu-Phe-Phe), to the formyl peptide receptors on the rabbit neutrophil was assessed by two approaches. A tritiated preparation of fMet-Leu-Phe-Phe was used for direct binding studies, whereas indirect studies comprised an assessment of the ability of the formyl tetrapeptide to competitively inhibit the binding of 35S-labeled formylmethionyl-leucyl-phenylalanine. These two approaches yielded analogous results. The formyl tetrapeptide fMet-Leu-Phe-Phe showed rapid and saturable binding to the same chemotactic receptors as the less potent formyl tripeptides with which it was compared. Its equilibrium-binding pattern, however, was different: fMet-Leu-Phe-Phe showed a homogeneous binding pattern, in contrast to the heterogeneity seen with the less potent compounds. The relative potencies for high-affinity binding of the two standard formyl tripeptides and fMet-Leu-Phe-Phe correlated well with their relative potencies for stimulating the biological response of degranulation; the relative potencies for low-affinity binding correlated less well.     

Regulation of alveolar macrophage production of chemoattractants by leukotriene B4 and prostaglandin E2.

Alveolar macrophages (AM) appear to influence the recruitment of neutrophils into the lung by the elaboration of both lipid and peptide chemotactic molecules for neutrophils. Little is known about the mechanisms that regulate production or release of chemotactic molecules by AM or the interaction between these classes of chemotactic molecules. We investigated the hypothesis that the lipid mediator leukotriene B4 (LTB4) has an in vitro regulatory action on the production of chemotactic proteins by AM. In these experiments, the chemotactic activity in AM culture supernatants was measured in a modified Boyden chamber. LTB4 treatment increased AM production of chemotactic activity in excess of what might be attributed to the amount of LTB4 measured in the culture supernatant after the incubation period. This effect was magnified by in vivo administration of endotoxin prior to AM harvesting. Pretreatment with LTB4 caused a sustained 250% increase in AM production of chemotactic activity, yet only negligible amounts of LTB4 were measured by gas chromatography/mass spectrometry in the LTB4-pretreated AM culture supernatants, indicating that LTB4 alone did not account for the chemotactic activity observed in our studies. A chemotactic peptide in LTB4-treated AM culture supernatant could be isolated and separated from LTB4 by molecular sieve chromatography. Purified column fractions contained 80% of the chemotactic activity of endotoxin-stimulated AM culture supernatant and had a molecular mass of 10,000 D. In contrast to LTB4, prostaglandin E2 (PGE2) suppressed chemotactic activity production by endotoxin-stimulated AM by 70%. Pretreatment with PGE2 was not effective; PGE2 had to be present in the AM culture medium during endotoxin exposure in order to exert a suppressive effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective inhibition of the C5a chemotactic cofactor function of the vitamin D binding protein by 1,25(OH)2 vitamin D3

The Vitamin D binding protein (DBP) is a multifunctional plasma protein that can significantly enhance the chemotactic response to complement fragment C5a. The chemotactic cofactor function of DBP requires cell surface binding in order to mediate this process. The goal of this study was to investigate the effect of ligating DBP with its two primary physiological ligands, Vitamin D and G-actin, on both binding to neutrophils and the ability to enhance chemotaxis to C5a. There was no difference in neutrophil binding between of the holo (bound) forms versus the apo (unbound) form of radioiodinated DBP, indicating that the cell binding region of DBP is likely distinct from the Vitamin D sterol and G-actin binding sites. Likewise, G-actin, 25(OH)D3, and G-actin plus 25(OH)D3 bound to DBP did not alter its capacity to enhance chemotaxis toward C5a. However, the active form of Vitamin D (1,25(OH)2D3) completely eliminated the chemotactic cofactor function of DBP. Dose-response curves demonstrated that as little as 1pM 1,25(OH)2D3 significantly inhibited chemotaxis enhancement. Moreover, at physiological concentrations 1,25(OH)2D3 needs to be bound to DBP to mediate the inhibitory effect. Neutrophil chemotaxis to optimal concentrations of C5a, formyl peptide, CXCL8 or leukotriene B4 was not altered by 1,25(OH)2D3, indicating that the active vitamin does not have a global inhibitory effect on neutrophil chemotaxis. Finally, inhibition of cell surface alkaline phosphatase (AP) with sodium orthovanadate completely reversed the inhibitory effect of 1,25(OH)2D3. These results indicate that the cell binding and co-chemotactic functions of DBP are not altered when the protein binds G-actin and/or Vitamin D. Furthermore, the co-chemotactic signal from DBP can be eliminated or counteracted by 1,25(OH)2D3.     

北京鸭红细胞膜G—蛋白与磷酯酶C的偶联及腺苷受体的调节

GDP、GTP和GTPγS激活北京鸭红细胞膜上的磷酯酶C,在10~(-7)～10~(-4)mol/L的范围内,使磷酸肌醇产量增加。其中GTPγS作用最强,分别使IP_2和IP_3增强550％和580％。GTP与GDP的效力相近,使IP_2增加140％和150％;IP_3增加230％和330％。A-嘌呤能受体激动剂6-N-cyclohe-xyladenosine(CHA)对抗ATP通过P-嘌呤能受体激活的磷酯酶C,使其活性下降60％。CHA的这种作用可被其特异拮抗剂8-cyclopentyl-1,3-dimethylxanthine对抗。以上结果表明鸭红细胞是研究受体、G-蛋白及磷酯酶C偶联的良好模型。表明不同嘌呤能受体在调节磷酯酶C中的相反作用。