Cytokines induce lymphocyte migration in vitro by direct,receptor-specific mechanisms

Several cytokines have been found to induce human peripheral blood lymphocyte (PBL) migration in vitro. The mechanisms involved are unclear, therefore experiments were carried out to determine whether PBL migration in response to selected cytokines is due to a direct effect, or to the generation of nonspecific secondary mediators, and whether migration is receptor specific. Purified human PBL were incubated with the lymphocyte chemotactic cytokines interleukin (IL)-1α and IL-8, followed after 30--60 min by addition of specific neutralizing monoclonal antibody. Supernatants of these mixtures were shown by subsequent assay to be devoid of PBL attractant activity, whereas positive control supernatants containing no antibody induced dilution-related migration. Addition of antibody to the high affinity IL-2 receptor abolished the potent attractant effect of IL-2 on PBL, but had little effect on responses to IL-1α and IL-8. These results demonstrate that the in vitro locomotor responses of human PBL to selected cytokines are due to direct, receptor-specific effects and are not dependent upon the generation of secondary mediator(s).     

Antigen-induced neutrophil chemotactic activity from sensitized lung

We have used the model of ovalbumin-sensitized guinea pigs to define the source of antigen-derived, high-molecular-weight neutrophil chemotactic activity (NCA). Incubating sensitized lung fragments with specific antigen (ovalbumin) but not irrelevant antigen (bovine serum albumin) or buffer resulted in the release of high-molecular-weight NCA and histamine, suggesting a lung mast cell origin for NCA. High-molecular-weight NCA accounted for greater than 90% of the NCA observed after antigen-lung incubation. The importance of high-molecular-weight NCA in recruitment of neutrophilic leukocytes to the site of allergic inflammatory reactions is emphasized.     

Eosinophil migration in response to three molecular species of platelet activating factor

Multiple molecular species of the eosinophil chemoattractant platelet activating factor (PAF) are produced as a result of inflammatory processes. We therefore compared the ability of three naturally occurring PAF species (C160, C180, and C181), which only varied at carbon 1, to induce eosinophil chemotaxis through naked 3-m pore polycarbonate filters. Timecourse experiments indicated that all species of PAF tested induced significant and equivalent eosinophil migration at 1 h which peaked at 2 h. Overall, the rank order of chemotactic potency for the PAF species was relatively equivalent. The specific PAF antagonist WEB 2086 inhibited eosinophil migration induced by all three PAF species equally. We conclude that the degree of PAF-induced eosinophil migration is not dependent upon the molecular species of PAF.accepted by G. W. CarterThis work was supported in part by a Veterans Administration Merit Review Award.     

Cytokines that regulate autoimmune responses

Autoimmune responses are controlled by complex regulatory circuits. Previous work has revealed that factors controlling autoimmunity can act both as potentiating and inhibitory agents, depending upon the site and timing of exposure. Recent advances in this complex field have at least partially uncovered the mechanism whereby these regulatory molecules participate in autoimmune processes. IL-12 production in the absence of infection may predispose to autoimmunity. IL-4 and transforming growth factor beta may suppress autoreactive T cells. Proinflammatory cytokines may ameliorate autoimmunity, dependent on the timing and level of production. In many cases, cytokines may act via antigen-presenting cells.     

Allergen extracts directly mobilize and activate human eosinophils

Allergic diseases are characterized by the presence of eosinophils, which are recruited to the affected tissues by chemoattractants produced by T cells, mast cells and epithelium. Our objective was to evaluate if allergens can directly activate human eosinophils. The capacity of purified allergen extracts to elicit eosinophil chemotaxis, respiratory burst, degranulation and up-regulation of the adhesion molecule complement receptor 3 (CR3) was determined in eosinophils isolated from healthy blood donors. Eosinophils stimulated with an extract from house dust mite (HDM) released the granule protein major basic protein (MBP) and up-regulated the surface expression of CR3. Cat allergen extracts also induced the up-regulation of CR3, but not the release of MBP; instead cat, as well as birch and grass allergens, elicited the release of eosinophil peroxidase (EPO). In addition, grass pollen extract caused the secretion of MBP. None of the allergens stimulated eosinophilic cationic protein release, nor production of free oxygen radicals. Both HDM and birch extracts were chemotactic for eosinophils. These findings establish that common aeroallergens can directly activate eosinophils in vitro. We propose that eosinophil activation in vivo is not exclusively mediated by cytokines and chemokines of the allergic inflammatory reaction, but could partly be the result of direct interaction between allergens and eosinophils.     

Modification of neutrophil chemotactic responsiveness during various inflammatory reactions

Various inflammatory reactions have been induced in order to examine the chemotactic response of polymorphonuclear leucocytes (PMN) collected under various experimental conditions. Cells were harvested from the pleural cavity of rats after the induction of three acute non specific inflammatory reactions and two immune reactions. The results obtained with two techniques of chemotactic assessment (agarose assay and Boyden chamber technique) demonstrated a variation of chemotactic response depending on the cell source and the chemoattractants used. Using agarose assay, we distinguished locomotor reactivity of PMN harvested after immune inflammatory reactions from that of PMN harvested after non immune inflammatory reactions. Chemokinetic and chemotactic responses to various chemoattractants were inhibited in the first case and not affected in the second. Using the Boyden chamber technique, inhibition of random or oriented migration of PMN harvested after immune inflammatory reactions after the injection of a non antigenic irritant such as calcium pyrophosphate crystals (CaPP) was also observed.     

IL-15 induces mast cell migration via a pertussis toxin-sensitive receptor

IL-15 induces proliferation, inhibits apoptosis and increases IL-4 production in murine mast cells. There is evidence that these activities are mediated via the uncharacterised receptor, IL-15R-X, rather than the classical three-chain IL-15 receptor. Effects of IL-15 on important aspects of mast cell biology, such as migration and degranulation, are unknown. We report that IL-15 induces migration of murine and human mast cells in a dose-dependent and biphasic manner, with peaks of migration occurring at approximately 10(-15) and approximately 10(-9) M. The potency of the response was similar to that induced by other well-established mast cell chemoattractants. Competition assays performed with murine and human mast cells indicate that both peaks of migration are due to chemotaxis. Pre-treatment of cells with pertussis toxin (PTX), a guanine nucleotide-binding regulatory protein (G-protein) inhibitor, resulted in complete inhibition of murine mast cell migration at approximately 10(-15) M IL-15, and human mast cell migration at approximately 10(-15) and approximately 10(-9) M. This demonstrates that murine and human mast cells express a PTX-sensitive receptor, activated in response to IL-15. Additionally, IL-15 did not induce degranulation in murine mast cells. Locally-produced IL-15 may contribute to mast cell recruitment during inflammatory responses, thereby acting as a linking cytokine between innate and adaptive arms of the immune system.     

哮喘中性粒细胞趋化因子性质的初步研究

测定哮喘发作和缓解期患者的中性粒细胞趋化因子活性,对中性粒细胞趋化因子(NCF)的理化性质进行初步研究。发现哮喘发作期NCA显著升高,RNCF对热较稳定,对蛋白酶敏感。脂溶性NCF活性仅占总NCF活性的4.2%。Sephadex G-200层析表明有两种NCF,一种分子量大于500000,另一种分子量约10000,前者是哮喘发作时的主要NCF。     

In vitro andin vivo impact of a new glycosphingolipid on neutrophils

A new water-soluble, orally absorbable de-N-acetyl-lysoganglioside (WILD20), breakdown product of the monosialoganglioside GM₁, was found to influence some parameters of neutrophil response to inflammation stimuli. Superoxide anion production appears inhibited, along with neutrophil killing properties. A block of both pathways of arachidonic acid cascade and PAF was also found, as well as neutrophil ICAM-1-mediated adhesion to endothelial cells. Of particular interest was the significant reduction of neutrophils observed at the site of inflammation, whichever agonist was used. The effects on neutrophil physiology found in normal or in pathological conditions, are in favour of a WILD20-related inhibitory effect on neutrophil contribution to inflammation.     

Chemotaxis of canine polymorphonuclear neutrophil granulocytes using the under-agarose method applied to glass microscope slides

The under-agarose chemotaxis assay method applied to glass microscope slides was optimised for canine polymorphonuclear granulocytes (PMN) with gelatin as protein supplement. Optimum chemotactic response occurred after approximately 135 min of incubation at 37 °C with 5 × 10⁵ cells/well and zymosan-activated serum (ZAS) as chemoattractant. Chemotactic (C) and random migration (R) were stated separately for each dog. Storage of blood samples for 90 min before isolation of PMN did not influence the C or R values (p > 0.05). The preanalyfcal variation was investigated and the proportion of variance, expressed as a percentage, due to each source for the C and R values, respectively, was determined: dog, 92%, 96%; blood sample, 0.4%, 0%; interslide, 3%, 2% and intraslide, 3%, 3%. The mean chemotactic responsiveness against ZAS and ZAS diluted 1:1 with RPMI 1640 (dZAS) was determined using 15 laboratory beagle dogs: C_ZAS = 0.67 mm (range: 0.25–2.33 mm), coefficient of variation (CV) = 24%, C_dZAS = 0.47 mm (range: 0.18–2.12 mm), CV = 22%. Also, the mean random migration was determined: 0.11 mm (range: 0.05–0.34 mm), CV = 25%. Neither age nor sex had any significant influence on chemotactic or random migration (p>0.05). The effect of heat-inactivated pooled canine serum (HI-serum) incorporated into agarose was examined and it was found that this had a significant stimulatory effect on both chemotactic and random migratory responses (p<0.05). Therefore, agarose with HI-serum is not useful when investigating the chemotactic function.     

Chemotaxis of canine polymorphonuclear neutrophil granulocytes using the under-agarose method applied to glass microscope slides

The under-agarose chemotaxis assay method applied to glass microscope slides was optimised for canine polymorphonuclear granulocytes (PMN) with gelatin as protein supplement. Optimum chemotactic response occurred after approximately 135 min of incubation at 37 °C with 5 × 10⁵ cells/well and zymosan-activated serum (ZAS) as chemoattractant. Chemotactic (C) and random migration (R) were stated separately for each dog. Storage of blood samples for 90 min before isolation of PMN did not influence the C or R values (p > 0.05). The preanalyfcal variation was investigated and the proportion of variance, expressed as a percentage, due to each source for the C and R values, respectively, was determined: dog, 92%, 96%; blood sample, 0.4%, 0%; interslide, 3%, 2% and intraslide, 3%, 3%. The mean chemotactic responsiveness against ZAS and ZAS diluted 1:1 with RPMI 1640 (dZAS) was determined using 15 laboratory beagle dogs: C_ZAS = 0.67 mm (range: 0.25–2.33 mm), coefficient of variation (CV) = 24%, C_dZAS = 0.47 mm (range: 0.18–2.12 mm), CV = 22%. Also, the mean random migration was determined: 0.11 mm (range: 0.05–0.34 mm), CV = 25%. Neither age nor sex had any significant influence on chemotactic or random migration (p>0.05). The effect of heat-inactivated pooled canine serum (HI-serum) incorporated into agarose was examined and it was found that this had a significant stimulatory effect on both chemotactic and random migratory responses (p<0.05). Therefore, agarose with HI-serum is not useful when investigating the chemotactic function.     

Cytokines and signal transduction

Cytokines are signaling molecules that coordinate cellular interactions in immune and hematopoietic systems. During the past 5 years many cytokines and their receptors have been identified and cloned. With a few exceptions, cytokine receptors do not contain any known signaling domains and therefore, in order to trigger a specific cellular response, new and unusual features of signaling pathways must be assumed. A major advance in the field has come with the discovery of new family of cytoplasmic protein tyrosine kinases that associate with occupied cytokine receptors and make them competent for intracellular signal generation. This review describes some general characteristics of cytokine signaling.Abbreviations CSF colony-stimulating factor - TGF transforming growth factor Correspondence to: J. M. Pfeilschifter     

Detection of C5a receptors on human eosinophils and inhibition of eosinophil effector functions by anti-C5a receptor (CD88) antibodies

Eosinophils and complement activation are reported to play a crucial role in the pathogenesis of connective tissue diseases. Depositions of antigens and antigen-antibody complexes lead to complement activation with the generation of anaphylatoxins, particularly C5a, which is thought to be responsible for the infiltration and activation of eosinophils in the tissue. Previous studies suggested that the eosinophil C5a receptor differs structurally from the receptor expressed on neutrophils. In this study, we investigated the expression and functional properties of C5a receptors on human eosinophils using the C5a receptor monoclonal antibody S5/1 (anti-CD88 mAb). Flow cytometric analysis demonstrated that the anti-CD88 mAb bound homogeneously on the surface of human eosinophils from nonatopic healthy donors. In addition, no subpopulations with respect to C5a receptor expression were identified in normodense or hypodense eosinophils of patients with hypereosinophilia. Pre-incubation of eosinophils with anti-CD88 specifically inhibited C5a-induced intracellular calcium concentration transients. C5a-induced chemotactic activity of eosinophils was significantly inhibited after pre-incubation of cells with anti-CD88 mAb in a dose-dependent manner. Furthermore, anti-CD88 mAb inhibited dose-dependently the release of reactive oxygen species by eosinophils following stimulation with C5a. Thus, the human eosinophil C5a receptor is homogeneously expressed on normal eosinophils from healthy donors as well as on hypodense and normodense eosinophil subpopulations from patients with hypereosinophilia. Based on the inhibitory effect of the S5/1 mAb on C5a-stimulated eosinophil effector functions, we conclude that a single C5a receptor type exists on human eosinophils. In addition, the inhibitory effect of the S5/1 mAb on C5a functions may enable a new experimental approach to the treatment of diseases that have been associated with C5a-mediated activation.     

哮喘患者痰嗜酸性粒细胞凋亡与IL-5及sIL-2R相关性的研究

目的：探讨哮喘患者痰液嗜酸性粒细胞凋亡(EOS)与IL-5和sIL-2R的关系。方法：选择哮喘患者(哮喘组)急性发作期和缓解期30例，20名健康人(正常组)作对照。分别采用ELISA法测定患者痰液白细胞介素5(IL-5)和可溶性白介素2受体(sIL-2R)，应用流式细胞仪检测痰液EOS凋亡。结果：哮喘患者急性发作期和缓解期痰液中IL-5和sIL-2R明显升高，EOS凋亡率增高，而正常组未发现EOS凋亡。急性发作期哮喘患者EOS凋亡率与IL-5呈明显的负相关，r为～0.78。结论：哮喘患者气道局部有EOS凋亡现象，并受到IL-5相互作用的调节。sIL-2R与EOS凋亡率无明显的相关性。     

抗炎6号、链霉素、氯霉素对大鼠中性粒细胞移动的影响

给大鼠连续三天每天一次静脉内注射中药抗炎6号(antiinflammatio No 6,AI6)4ml可明显地促进其中性粒细胞(neutrophil granulocyte,NG)的趋化性(chemotaxis,Cht)。连续三天每天一次肌肉内注射链霉素(streptomycin,SM)和氯霉素(chloromycetin,CM)各50mg,可明显地抑制大鼠NG的Cht。如果连续三天每天一次肌肉内注射SM和CM各50mg,同时又每天一次静脉内注射AI6 4ml,则可见AI6不仅能完全对消SM和CM对NG Cht的抑制,而且还能进一步促进Cht。     

Prophylactic simvastatin increased survival during endotoxemia and inhibited granulocyte trafficking in a cell-intrinsic manner

Cross sectional studies have shown that statin-users have improved odds of surviving severe sepsis. Meanwhile controlled clinical trials failed to demonstrate improved sepsis survival with acute statin administration following hospitalization. Here, a lethal murine peritoneal lipopolysaccharide (LPS) endotoxemia model was used to assess the efficacy of chronic versus acute simvastatin on survival. Mirroring clinical observations, chronic but not acute treatment with simvastatin significantly increased survival. At a pre-mortality time point in LPS-treated mice, chronic simvastatin suppressed granulocyte trafficking in to the lungs and peritoneum without otherwise suppressing emergency myelopoiesis, myeloid cells in circulation, or inflammatory cytokines. Chronic simvastatin treatment significantly downregulated inflammatory chemokine gene signature in the lungs of LPS-treated mice. Thus, it was unclear if simvastatin was inhibiting granulocyte chemotaxis in a cell intrinsic or extrinsic manner. Adoptive transfer of fluorescently labeled granulocytes from statin and vehicle treated mice into LPS-treated mice showed that simvastatin inhibited lung-granulocyte trafficking in a cell intrinsic manner. Congruent with this, chemotaxis experiments using in vitro macrophages and ex vivo granulocytes demonstrated that simvastatin inhibited chemotaxis in a cell-intrinsic manner. Collectively, chronic but not acute simvastatin treatment improved survival in murine endotoxemia, and this was associated with cell-intrinsic inhibition of granulocyte chemotaxis.     

Neutrophil Migration in Opposing Chemoattractant Gradients Using Microfluidic Chemotaxis Devices

Neutrophils migrating in tissue respond to complex overlapping signals generated by a variety of chemotactic factors (CFs). Previous studies suggested a hierarchy between bacteria-derived CFs and host-derived CFs but could not differentiate neutrophil response to potentially equal host-derived CFs (IL-8 and LTB₄). This paper reports neutrophil migration in conflicting gradients of IL-8 and LTB₄ using a microfluidic chemotaxis device that can generate stable and well-defined gradients. We quantitatively characterized the movement of cells from time-lapse images. Neutrophils migrate more efficiently toward single IL-8 gradients than single LTB₄ gradients as measured by the effective chemotactic index (ECI). In opposing gradients of IL-8 and LTB₄, neutrophils show obvious chemotaxis toward a distant gradient, consistent with previous reports. When an opposing gradient of LTB₄ is present, neutrophils show less effective chemotaxis toward IL-8 than when they are in a gradient of IL-8 alone. In contrast, the chemotactic response of neutrophils to LTB₄ is not reduced in opposing gradients as compared to that in a single LTB₄ gradient. These results indicate that the presence of one host-derived CF modifies the response of neutrophils to a second CF suggesting a subtle hierarchy between them.This revised version was published online in May 2005 with corrected author affiliations     

Lipocalin‐2 ameliorates granulocyte functionality

Attraction of neutrophils to sites of infection or tissue injury is an essential prerequisite for an efficient innate immune response. Herein, we provide novel evidence that the antimicrobial protein, neutrophil gelatinase associated lipocalin (24p3 or lipocalin‐2, Lcn2) is a central regulator of this process. Lcn2 is produced by several cell types but high amounts are released by neutrophils. Using human and murine neutrophils, we found that the addition of recombinant Lcn2 significantly stimulated their migration, which was independent of IL‐8/keratinocyte chemokine formation. Mechanistically, this could be traced back to Lcn2‐mediated changes of Erk1/2 signaling. Accordingly, the i.p. injection of Lcn2 into C57BL/6 mice stimulated the mobilization of neutrophils while we found a significantly reduced neutrophil chemotactic activity of cells obtained from Lcn2 KO mice. This observation transmitted to a reduced accumulation of neutrophils in intra‐dermal lesions infected with Salmonella typhimurium in Lcn2 KO mice as compared to WT mice. This was not only due to a reduced chemotaxis but also to an impaired cellular adhesion of neutrophils in the absence of Lcn2. We herein describe a novel role of Lcn2 as an important paracrine chemoattractant and an indispensable factor for neutrophil function in inflammation.     

The role of the LTB4-BLT1 axis in chemotactic gradient sensing and directed leukocyte migration

Directed leukocyte migration is a hallmark of inflammatory immune responses. Leukotrienes are derived from arachidonic acid and represent a class of potent lipid mediators of leukocyte migration. In this review, we summarize the essential steps leading to the production of LTB₄ in leukocytes. We discuss the recent findings on the exosomal packaging and transport of LTB₄ in the context of chemotactic gradients formation and regulation of leukocyte recruitment. We also discuss the dynamic roles of the LTB₄ receptors, BLT1 and BLT2, in mediating chemotactic signaling in leukocytes and contrast them to other structurally related leukotrienes that bind to distinct GPCRs. Finally, we highlight the specific roles of the LTB₄-BLT1 axis in mediating signal-relay between chemotaxing neutrophils and its potential contribution to a wide variety of inflammatory conditions including tumor progression and metastasis, where LTB₄ is emerging as a key signaling component.