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S Nourshargh

Publications and source records attributed to S Nourshargh.

At least 37 records · Page 2Linked to original sources
PubMed1995

Molecular and cellular interactions mediating granulocyte accumulation in vivo.

An inflammatory response, either beneficial in host defence or detrimental resulting in an inflammatory disease, is associated with alterations in vascular tone and blood flow, enhanced vascular permeability to macromolecules and the extravasation of leucocytes from the vascular lumen into extravascular tissue. The adhesive interaction of granulocytes with venular endothelial cells is an essential step in the process of granulocyte accumulation at sites of inflammation in vivo. Recent advances made in the field of chemokines and adhesion molecules have led to a better understanding of the molecular events mediating this important component of the inflammatory response. The defined molecular interactions that mediate and regulate these events, in the process of neutrophil accumulation, will be discussed in this article. Recently, the eosinophil has come into focus because of its prominence in allergic reactions. This cell will be discussed in comparison.

Animals↗
PubMed1995

Substance P-induced inflammatory responses in guinea-pig skin: the effect of specific NK1 receptor antagonists and the role of endogenous mediators.

1. The sensory neuropeptide substance P (SP), when released from sensory nerves, has been implicated in the development of neurogenic inflammation. In the present study, using an in vivo model system, we have characterized and investigated the mechanisms underlying SP-induced leukocyte accumulation and oedema formation in the guinea-pig. 2. Intradermally injected SP (i.d., 10(-13) - 10(-9) mol per site), induced a dose- and time-dependent accumulation of 111In-neutrophils, 111In-eosinophils and oedema formation as measured by the local accumulation of i.v. injected 125I-albumin. The leukocyte accumulation evoked by SP was significant at 10(-10) and 10(-9) mol per site, whereas oedema formation was significant at the lowest dose tested (10(-13) mol per site). 3. The NK1 receptor antagonists, CP-96,345 (1 mg kg-1, i.v.) and RP-67,580 (10 micrograms per site, i.d.), significantly attenuated the oedema formation induced by the lower doses of SP. Oedema formation and leukocyte accumulation induced by 10(-9) mol per site SP were unaffected by either antagonist. 4. SP-elicited responses were not significantly affected by the platelet activating factor (PAF) receptor antagonist, UK-74,505 (2.5 mg kg-1, i.v.) or the H1 histamine receptor antagonist, chlorpheniramine (10(-8) mol per site, i.d.). However, the 111In-eosinophil accumulation, but not the 111In-neutrophil accumulation or oedema formation, induced by SP was significantly inhibited by the specific 5-lipoxygenase (5-LO) inhibitor, ZM-230,487 (10(-8) mol per site, i.d.). 5. The accumulation of both 111 In-neutrophils and 111 In-eosinophils induced by SP was abolished in guinea-pigs treated i.v. with an anti-CD18 monoclonal antibody 6.5E F(ab')2 (2.5 mg kg-1). The oedema response was unaffected in these animals.6. These results suggest that SP-induced inflammatory events may be mediated via two mechanisms involving NK1 receptor-dependent and independent pathways. Oedema formation induced by the lower doses of SP may be mediated via the direct activation of NK1 receptors whilst, at higher doses, oedema formation and leukocyte accumulation may be mediated via the release of secondary mediators, possibly mast cell derived, with 5-LO products playing an important role in the leukocyte infiltration. The leukocyte accumulation, but not the oedema induced by SP, is dependent on the expression of the CD18antigen on leukocytes.

Animals↗
PubMed1995

Lung eosinophilia is dependent on IL-5 and the adhesion molecules CD18 and VLA-4, in a guinea-pig model.

Blood and tissue-eosinophilia is a characteristic feature of a number of disease states. In experimental animals, the intravenous injection of parasitic larvae induces a profound eosinophilia that can be mimicked by the intravenous injection of Sephadex particles. In the present study, this procedure was used to investigate the mechanisms involved in the development of lung eosinophilia in a guinea-pig model. Intravenous administration of Sephadex particles to guinea-pigs resulted in a significant increase in the influx of eosinophils in the airways and in lung tissue eosinophil peroxidase (EPO) activity (at t = 24 hr). An anti-interleukin-5 (IL-5) monoclonal antibody (mAb) totally inhibited the eosinophilia in the airways and significantly reduced the lung tissue EPO activity. The concomitant accumulation of neutrophils and mononuclear cells, however, was not affected by this treatment. Monoclonal antibodies to VLA-4 and CD18 caused 58% and 62% suppression of eosinophilia in the bronchoalveolar lavage (BAL), respectively, whilst having no effect on lung tissue EPO activity. Co-administration of the two mAb resulted in total inhibition of eosinophil accumulation into BAL and significant suppression of lung tissue EPO activity (55% inhibition). This procedure also resulted in 72% inhibition of mononuclear cell influx and 68% inhibition of neutrophil influx in the BAL, the latter effect being entirely due to the actions of the anti-CD18 mAb. The results of this study indicate for the first time a requirement for IL-5 in the development of lung eosinophilia in this model. Further, it is clear that both the molecules VLA-4 and CD18 contribute to the development of this response and that maximal inhibition of lung eosinophilia is achieved only when the two adhesion pathways are simultaneously blocked.

Animals↗
PubMed1995

LPS-induced 111In-eosinophil accumulation in guinea-pig skin: evidence for a role for TNF-alpha.

Lipopolysaccharide (LPS) is a major component of the cell wall of Gram-negative bacteria with powerful pro-inflammatory activities. Although the mechanisms involved in LPS-induced neutrophil accumulation have been studied extensively, few reports have focused on the effects of LPS on eosinophil infiltration. In this study we have used an in vivo model of local 111In-eosinophil accumulation in the guinea-pig to investigate the mechanisms of LPS-induced eosinophilia. Using a 4-hr in vivo test period, the intradermal injection of LPS (50-1000 ng/site) led to a marked and dose-dependent accumulation of 111In-eosinophils into guinea-pig skin sites. Time-course experiments revealed that this cell infiltration was delayed in onset, becoming significant 1 hr after the intradermal administration of LPS. The slow development of the response and its sensitivity to the locally administered protein synthesis inhibitor, actinomycin D, suggested that the LPS-induced 111In-eosinophil accumulation in vivo is mediated by the generation of de novo proteins. The intravenous pretreatment of guinea-pigs with a soluble tumour necrosis factor-alpha (TNF-alpha) receptor fusion protein (TNFR-IgG, 1 mg/kg), potently inhibited the 111In-eosinophil accumulation induced by LPS. Our results demonstrate that LPS can induce 111In-eosinophil accumulation in vivo in guinea-pig skin, and that this process is mediated by TNF-alpha.

Animals↗
PubMed1994

Differential effects of the PAF receptor antagonist UK-74,505 on neutrophil and eosinophil accumulation in guinea-pig skin.

1. The effect of the dihydropyridine, platelet activating factor (PAF) receptor antagonist, UK-74,505, on leucocyte accumulation and oedema formation in guinea-pig skin was investigated. The inflammatory reactions studied were elicited by exogenous mediators, a passive cutaneous anaphylactic (PCA) reaction and zymosan particles. 2. Leucocyte accumulation and oedema formation were measured as the local accumulation of i.v. administered 111In-labelled neutrophils or eosinophils together with 125I-labelled albumin. UK-74,505 was either administered i.v. or used to pretreat the radiolabelled leucocytes in vitro prior to their last wash and injection into recipient animals. 3. In vitro, UK-74,505 inhibited PAF-induced elevations in cytoplasmic levels of Ca2+ ([Ca2+]i) in fura-2-loaded guinea-pig neutrophils and eosinophils with IC50 values of 10(-9) M and 7 x 10(-9) M respectively. Neutrophils and eosinophils pretreated with 10(-7) M and 10(-6) M UK-74,505 respectively, and maintained at 37 degrees C, were unresponsive to PAF for the 4 h period investigated. 4. In vivo, using 2 h test periods, i.v. UK-74,505 (0.5 and 2.5 mg kg-1) inhibited the accumulation of 111In-neutrophils, 111In-eosinophils and oedema formation induced by intradermal PAF, but had no effect on responses elicited by leukotriene B4 (LTB4) and zymosan-activated plasma (ZAP, used as a source of C5a des Arg). UK-74,505 (2.5 mg kg-1) was also without an effect on response induced by a PCA reaction but significantly suppressed the 111In-eosinophil accumulation following the intradermal administration of zymosan particles. The 111In-neutrophil accumulation induced by zymosan particles was not, however, affected by UK-74,505. 5. In a second series of in vivo experiments, "'In-leucocytes were pretreated in vitro with UK-74,505 prior to their last wash and injection into recipient animals. Radiolabelled neutrophils, and eosinophils were pretreated with 10-7 M and 10-6 M UK-74,505 respectively, concentrations previously shown to block the leucocyte responses to PAF in vitro for up to 4 h. The in vitro pretreatment of the cells with the PAF antagonist, whilst not affecting the responses to intradermally-injected PAF, suppressed the"'In-eosinophil accumulation response induced by zymosan particles.6. The results of this study indicate that PAF is not involved in neutrophil accumulation, eosinophil accumulation and oedema formation induced by LTB4, ZAP and a PCA reaction. Endogenous PAF does, however, appear to have a role in zymosan-induced eosinophil accumulation but not neutrophil accumulation, suggesting the existence of different inflammatory pathways in the induction of neutrophil and eosinophil accumulation in vivo. Furthermore, while leucocyte accumulation induced by exogenous PAF does not appear to involve leucocyte PAF receptors, the mechanism by which endogenous PAF mediates the zymosan-induced eosinophil accumulation appears dependent on the expression of PAF receptors on eosinophils.

Animals↗
PubMed1993

A monoclonal antibody recognizing very late activation antigen-4 inhibits eosinophil accumulation in vivo.

Using an in vivo test system, the role of the beta 1 integrin very late activation antigen-4 (VLA-4) in eosinophil accumulation in allergic and nonallergic inflammatory reactions was investigated. Eosinophil infiltration and edema formation were measured as the local accumulation of intravenously injected 111In-labeled eosinophils and 125I-human serum albumin. The inflammatory reactions investigated were a passive cutaneous anaphylaxis (PCA) reaction and responses elicited by intradermal soluble inflammatory mediators (platelet-activating factor, leukotriene B4, C5a des Arg), arachidonic acid, and zymosan particles. The in vitro pretreatment of 111In-eosinophils with the anti-VLA-4 monoclonal antibody (mAb) HP1/2, which crossreacts with guinea pig eosinophils, suppressed eosinophil accumulation in all the inflammatory reactions investigated. Eosinophil accumulation was inhibited to the same extent when mAb HP1/2 was administered intravenously. It is interesting that HP1/2 had no effect on stimulated edema formation. These results suggest a role for VLA-4 in eosinophil accumulation in vivo and indicate a dissociation between the inflammatory events of eosinophil accumulation and edema formation.

Animals↗
PubMed1993

Effect of dexamethasone on neutrophil accumulation and oedema formation in rabbit skin: an investigation of site of action.

1. The anti-inflammatory actions of dexamethasone on vascular and leukocyte responses in rabbit skin were investigated. 2. Neutrophil accumulation and oedema formation were simultaneously measured as the local accumulation of i.v. administered 111In-labelled neutrophils and 125I-labelled albumin. Systemically administered dexamethasone (3 mg kg-1) inhibited neutrophil accumulation induced by i.d. zymosan activated plasma (ZAP), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and leukotriene B4 (LTB4) when co-injected with prostaglandin E2 (PGE2). Dexamethasone also inhibited oedema formation elicited by these stimuli and the responses induced by i.d. platelet activating factor (PAF)+PGE2 and bradykinin (BK)+PGE2. 3. Intradermal dexamethasone (2 x 10(-10) mol per site) but not indomethacin (10(-8) mol per site) inhibited oedema formation induced by i.d. ZAP+PGE2 and BK+PGE2. This inhibitory effect of dexamethasone was significant only with pretreatment periods of 4 h, shorter pretreatment periods resulting in greatly reduced effects. Intradermal dexamethasone had no effect on neutrophil accumulation induced by ZAP+PGE2. 4. Intradermal dexamethasone (2 x 10(-10) mol per site) had no effect on increase in blood flow induced by PGE2 as measured by 133Xenon clearance. 5. The accumulation of neutrophils isolated from donor rabbits pretreated with i.v. saline or dexamethasone (3 mg kg-1) was investigated in untreated recipient rabbits. The accumulation of neutrophils, induced by ZAP+PGE2, FMLP+PGE2 and LTB4+PGE2, from dexamethasone-pretreated donors was significantly smaller than the accumulation of neutrophils from saline-pretreated donors. 6. The results of this study suggest that dexamethasone can have a direct effect on vascular endothelial cells resulting in an inhibition of oedema formation. 7. Neutrophil accumulation can be inhibited by an effect of dexamethasone on the neutrophil itself or on the vascular endothelium. These results indicate that at least part of the inhibitory effect is on the circulating neutrophil induced by dexamethasone or a dexamethasone-induced product.

Animals↗
PubMed1993

Role of platelet-activating factor (PAF) in platelet accumulation in rabbit skin: effect of the novel long-acting PAF antagonist, UK-74,505.

1. The contribution of platelet-activating factor (PAF) to platelet deposition and oedema formation induced by exogenous soluble mediators, zymosan particles and associated with a reversed passive Arthus (RPA) reaction in rabbit skin was investigated by use of a novel long-acting PAF receptor antagonist, UK-74,505. 2. Oedema formation and platelet accumulation were simultaneously measured by i.v. injection of [125I]-albumin and 111In-labelled rabbit platelets. UK-74,505 was either administered i.v. or used to pretreat radiolabelled platelets in vitro before their injection into recipient animals. Platelets pretreated with UK-74,505 were also labelled with the fluorescent calcium indicator, Fura-2, to assess their ex vivo reactivity to PAF at the end of the in vivo experiment. 3. UK-74,505 (0.5 mg kg-1), administered i.v., inhibited PAF-induced oedema formation, but did not affect oedema induced by zymosan particles, bradykinin (BK), histamine, formyl-methionyl-leucylphenylalanine (FMLP), zymosan-activated plasma (ZAP, as a source of C5a des Arg), leukotriene B4 (LTB4) or interleukin-8 (IL-8). 4. UK-74,505, administered i.v. also suppressed the small platelet accumulation induced by exogenous PAF, but had no effect on accumulation induced by IL-8 or ZAP. Although oedema induced by zymosan was not affected by i.v. UK-74,505, zymosan-induced platelet accumulation was significantly attenuated by the antagonist. 5. The RPA reaction in rabbit skin was associated with marked oedema formation and platelet accumulation which were both inhibited by i.v. UK-74,505. 6. In vitro, UK-74,505 inhibited aggregation and the increase in intracellular calcium concentration induced by PAF in rabbit washed platelets in a concentration-dependent manner (IC50 = 1.6 x 10-8 M and 1.1 x 10-8 M, respectively). Platelets pretreated with 10-6 M UK-74,505, and maintained at 37 degrees C,were unresponsive to PAF, whilst responding normally to thrombin, for up to 4 h.7. In a second series of in vivo experiments, platelets were labelled with 111In and loaded with Fura-2.The platelets were then pretreated with 10-6 M UK-74,505, washed, and injected into recipient rabbits.These platelets, prepared from blood samples taken at the end of the in vivo experiments, exhibited an 80% reduction in their response to PAF as measured ex vivo with Fura-2. However, in contrast to the effects of i.v. UK-74,505, platelets pretreated with the antagonist did accumulate effectively in the RPA reaction, a significant reduction only being observed in responses at the lowest antibody dose. In addition, pretreatment of platelets had no effect on the small platelet accumulation induced by PAF.8. These results suggest that PAF is an important mediator of oedema formation and platelet accumulation in the RPA reaction in rabbit skin. However, they question the role of PAF receptors on platelets in this model. The results also indicate that PAF may be involved in platelet accumulation induced by zymosan in rabbit skin.

Animals↗
PubMed1993

Mechanisms of neutrophil and eosinophil accumulation in vivo.

The accumulation of leukocytes into tissues is a characteristic feature of inflammatory reactions. This process is triggered by chemical signals generated in a tissue in response to an inflammatory stimulus e.g., invading microbes, other foreign organisms, allergens, or damaged tissue cells. The mechanisms involved in neutrophil and eosinophil accumulation in vivo are complex and dependent on an initial interaction between the leukocytes and the microvascular endothelial cells. This response is regulated by the coordinated expression and/or activation of leukocyte and endothelial cell adhesion molecules. The precise mechanisms that control the selective accumulation of eosinophils, as opposed to neutrophils, in certain inflammatory reactions (e.g., in IgE-mediated allergic reactions) remain unclear. This may be explained partly by the generation of eosinophil-specific inflammatory mediators and activation of selective adhesion pathways such as the VLA-4/VCAM-1 interaction. Although the neutrophil and eosinophil have distinct roles in host defense, they have been implicated in the pathogenesis of a number of inflammatory disorders. Thus, a better understanding of the events mediating and regulating neutrophil and eosinophil accumulation in vivo will be of considerable value in the development of therapeutic strategies for inflammatory disease states.

Animals↗
PubMed1992

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

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

Animals↗
PubMed1992

Comparative responses of human and rabbit interleukin-1 in vivo: effect of a recombinant interleukin-1 receptor antagonist.

The ability of recombinant human and rabbit interleukin-1 alpha (IL-1 alpha) in inducing inflammatory responses in rabbit skin were compared. Intradermal (i.d.) injections of recombinant human IL-1 alpha and recombinant rabbit IL-1 alpha induced intense accumulation of 111In-labelled neutrophils which was dependent on the dose of the cytokines administered. Both forms of IL-1 alpha induced very small levels of plasma protein leakage. Co-injection of the cytokines with the mRNA synthesis inhibitor actinomycin D (Act D) attenuated the number of neutrophils accumulating in response to both human and rabbit forms of IL-1 alpha. Local injection of a recombinant human IL-1 receptor antagonist (IL-1Ra) caused a dose-dependent inhibition of local inflammatory responses initiated by human and rabbit IL-1 alpha s well as rabbit IL-1 beta indicating the species cross-reactivity of the antagonist. IL-1Ra was selective for IL-1 in rabbit skin, as responses induced by C5ades Arg and formyl-methionyl-leucyl-phenylalanine (FMLP) were not inhibited. IL-1Ra significantly inhibited the IL-1-induced neutrophil accumulation only when co-injected with the cytokine. The local administration of the antagonist 30 min after rabbit IL-1 alpha failed to inhibit the inflammatory response. These results suggest that the in vivo events leading to the accumulation of neutrophils in response to IL-1 alpha are rapidly initiated.

Animals↗
PubMed1991

Mechanism of action of platelet activating factor in the pulmonary circulation: an investigation using a novel isotopic system in rabbit isolated lung.

1. Rabbit isolated lungs were perfused via the pulmonary artery with Tyrode solution containing 4.5% Ficoll and 0.1% bovine serum albumin at a constant rate of 20 ml min-1. Lung perfusate was drawn for alternating 5 min periods from two reservoirs, one containing 125I-albumin and the other unlabelled albumin to wash out the intravascular label. Microvascular 125I-albumin leakage was determined from the count remaining at the end of the washout phase with an external gamma scintillation probe. In addition, perfusion pressure was monitored continuously. Each experiment comprised 6 cycles over a total period of 60 min. 2. Infusion of platelet activating factor (PAF, 3 nmol min-1 for 10 min) resulted in microvascular 125I-albumin leakage, whereas lyso-PAF was without effect. During PAF infusions there was also an increase in perfusion pressure. Both the permeability and pressor effects of PAF were inhibited by the PAF antagonist L-652731. 3. Infusion of the thromboxane analogue U-46619 (0.6 nmol min-1 for 10 min) caused an increase in perfusion pressure but protein accumulation was not significantly different from that observed with control infusions. 4. Bolus injections of PAF (1 nmol) caused increases in perfusion pressure which were reduced by indomethacin, dazmegrel and BW 755C. Bolus injections of PAF, repeated at 30 min intervals caused reproducible pressor responses; however, repeated injections at 60 min intervals resulted in augmented responses. This augmentation did not occur in the presence of indomethacin. 5. Retrograde perfusion of PAF via the pulmonary vein induced increased perfusion pressure and microvascular 125I-albumin leakage. The observed increase in leakage when compared with forward perfusion suggests that PAF produces predominantly arteriolar constriction i.e. proximal to the site of leakage during forward perfusion. 6. These results indicate that PAF is a vasoconstrictor in the rabbit pulmonary circulation and augmented responses occur with repeated injections at 60 min intervals. Cyclo-oxygenase inhibition abolished this vascular hyperresponsiveness induced by PAF. PAF also caused protein accumulation in the lungs. Both these actions of PAF appear to be receptor-mediated because they were inhibited by PAF antagonists. Another pulmonary vasoconstrictor, U-46619 did not cause protein accumulation suggesting that the extravasation of protein with PAF is not merely secondary to changes in vascular tone.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1991

The accumulation of 111In-eosinophils induced by inflammatory mediators, in vivo.

Eosinophils are implicated in the pathogenesis of a variety of allergic inflammatory diseases such as asthma. Several substances have been shown to be chemotactic for eosinophils in vitro, but the inflammatory mediators involved in the accumulation of eosinophils in vivo are as yet unidentified. In this study we have developed a system to measure the accumulation of 111In-eosinophils in guinea-pig skin in vivo. Horse serum-induced guinea-pig peritoneal eosinophils were radiolabelled with 111In and injected intravenously into recipient animals. 125I-albumin was also injected intravenously in order to measure local oedema formation simultaneously. A range of putative mediators was injected intradermally and responses measured for up to 2 hr. Of the mediators tested, guinea-pig C5a des Arg in zymosan-activated plasma was the most active. Recombinant human C5a (rHC5a) was also highly active, but less than the guinea-pig material. C5a des Arg in maximally activated plasma induced a 1500% increase in eosinophil accumulation, while rHC5a (10(-10) mol dose) induced a 600% increase. Platelet-activating factor (PAF) and leukotriene B4 (LTB4) were also tested for comparison. With respect to 111In-eosinophil accumulation, the order of potency of the mediators tested was as follows: guinea-pig C5a des Arg greater than LTB4 greater than PAF. In contrast, the order of potency of the mediators with respect to oedema formation was: PAF greater than guinea-pig C5a des Arg greater than LTB4. The techniques described will facilitate analysis of the mechanisms involved in eosinophil accumulation in defined inflammatory reactions.

Albumins↗
PubMed1990

Evidence that a receptor-operated event on the neutrophil mediates neutrophil accumulation in vivo. Pretreatment of 111In-neutrophils with pertussis toxin in vitro inhibits their accumulation in vivo.

The role of neutrophil chemoattractant receptors in neutrophil stimulation in vitro is well established, however, the precise mechanisms underlying local neutrophil accumulation at inflammatory sites in vivo have not been defined. A fundamental question that remains open is whether chemoattractants act on the endothelial cell or the neutrophil to initiate the process of neutrophil migration in vivo. To address this question we have investigated whether neutrophil accumulation in vivo can occur if chemoattractant receptor occupancy is uncoupled from neutrophil stimulation. For this purpose we have used pertussis toxin (PT) as the pharmacologic tool. We have investigated the effect of in vitro pretreatment of rabbit neutrophils with PT on their responses in vitro and on their accumulation in vivo. Pretreatment of rabbit neutrophils with PT inhibited FMLP- and C5a-, but not PMA- induced increases in CD18 expression, neutrophil adherence, and degranulation in vitro. This pretreatment procedure with PT inhibited the accumulation of radiolabeled neutrophils in vivo in response to intradermally injected FMLP, C5a, C5a des Arg, leukotriene B4, IL-8, and zymosan in rabbit skin. Further, in contrast to the in vitro results, PT inhibited the PMA-induced 111In-neutrophil accumulation in vivo. Interestingly, pretreatment of neutrophils with PT also inhibited accumulation in response to intradermally injected IL-1, despite the reports that IL-1 lacks neutrophil chemoattractant activity in vitro. Although the experimental techniques used cannot distinguish the different stages of neutrophil migration involved, these results suggest that the accumulation of neutrophils induced by local extravascular chemoattractants in vivo depends on a pertussis toxin-sensitive receptor operated event on the neutrophil itself. Further, PMA and IL-1 may release secondary chemoattractants in vivo.

Animals↗
PubMed1989

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

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

Animals↗
PubMed1987

Divergent effects of co-carcinogenic phorbol esters and a synthetic diacylglycerol on human neutrophil chemokinesis and granular enzyme secretion.

The effects of two co-carcinogenic phorbol esters (phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBu] and a synthetic diacylglycerol (OAG, 1-oleoyl-2-acetyl-glycerol), which all stimulate protein kinase C, were compared with two inactive phorbol compounds (4 alpha-phorbol and 4 alpha-phorbol didecanoate (4 alpha-PDD)) on three functional properties of stimulated human polymorphonuclear leukocytes (PMNs): release of granular enzymes lysozyme and beta-glucuronidase, chemokinesis, and changes in cytoplasmic free calcium [Ca2+]i. PMA, PDBu and the diacylglycerol, OAG, all caused a dose-dependent and slow (max by 15 min) release of small amounts of lysozyme with much less beta-glucuronidase and no release of cytoplasmic lactate dehydrogenase. Release was unaffected by removal of extracellular Ca2+. PMA, PDBu and OAG inhibited random movement of the cells, did not cause chemokinesis and induced a slow reduction in the basal [Ca2+]i, as measured by the quin-2 method. PMA, PDBu and OAG increased the capacity of five independently-acting stimulants (N-formyl-Met-Leu-Phe, leukotriene B4, C5a des-Arg, platelet activating factor and A23187) to cause release of lysozyme and beta-glucuronidase but strongly inhibited PMN chemokinesis induced by the same five agents and reduced the stimulant-induced increases in [Ca2+]i. PMA was always more potent than PDBu and much more potent than OAG in eliciting these stimulatory or inhibitory effects on human PMNs. In all tests, 4 alpha-phorbol and 4 alpha-PDD were inactive. The results confirm that stimulation of the diacylglycerol/protein kinase C system in human PMN, either by active phorbol esters or the synthetic diacylglycerol, causes bidirectional effects on human PMN function. In particular, activation of the C-kinase causes inhibition of stimulated neutrophil motility, whereas the secretory functions of the cells are enhanced.

Calcium↗