Modulation of eosinophil chemotaxis and eosinophil chemotactic factor release by concanavalin A

Concanavalin A (Con A) is not a chemotactic factor for guinea pig eosinophil leukocytes. It does, however, modulate the response of cells to neutrophil-derived eosinophil chemotactic factor (ECF) or to C5a. This effect occurs by a direct interaction of Con A with the target cell and is reversible by appropriate doses of alpha-methyl-D-mannoside (alpha-MM). The inhibition of chemotaxis at high doses of Con A is due to agglutination of the cells on top of the filter. The mechanism of enhancement at low doses is not understood, but several possible explanations are explored. Careful definition of the effect of Con A on eosinophil chemotaxis makes it possible to exclude an effect of Con A on ECF secretion by neutrophils.     

Inhibition of human eosinophil chemotaxis by IgA paraproteins

Sera from patients with IgA myeloma inhibit normal human eosinophil chemotaxis. No correlation was noted between inhibition and the absolute concentration of IgA or - light-chain type. Eosinophil chemotactic inhibitory activity was associated with isolated IgA paraproteins and was found to be cell directed and stable at 56° C. Pepsin digestion of IgA paraproteins resulted in loss of both IgA Fc fragment and eosinophil chemotactic inhibitory activity. Polymeric IgA accounted for most of the inhibitory activity as evidenced by sucrose density gradient centrifugation studies and a loss of inhibitory activity following dithiothrietol reduction and iodoacetamide alkylation which converted polymeric IgA to monomeric IgA. Comparative studies with neutrophils showed that both neutrophil and eosinophil chemotaxis and chemokinesis were effectively inhibited by IgA paraproteins. The mechanisms of suppression of eosinophil and neutrophil chemotaxis by IgA paraproteins appear to be similar and possibly may involve a membrane receptor for IgA.     

Inactivation of polymorphonuclear-neutrophil derived eosinophil chemotactic factor by human serum.

A low-molecular-weight eosinophil chemotactic factor (ECF) can be released from human neutrophils on stimulation with the calcium ionophore (A 23187) and during phagocytosis. The presence of human serum suppresses the chemotaxis of eosinophils by ECF significantly. The inhibition is not cell-directed but affects the ECF either by binding or enzymatic cleavage. The inactivators belong to a heterogeneous class of serum components in the 19s, 7s and 4s molecular weight range. Highly purified human IgM and IgA, but neither IgG nor human serum albumin inhibit eosinophil chemotaxis.     

Inhibition of eosinophil chemotaxis by a new antiallergic compound (cetirizine)

The in vivo inhibitory effect of a new antiallergic, anti-H1 drug, cetirizine, on eosinophil attraction at skin sites challenged with various stimuli has been recently suggested. In the present work, we confirmed that this molecule, at therapeutical concentration, has a potent inhibitory action on eosinophil response to different chemoattractant mediators such as platelet-activating factor (PAF acether) and N-formyl methionyl leucyl phenyl alanyl in vitro. Another anti-H1 drug, polaramine, did not show this effect at the same concentration. These findings suggest that cetirizine in addition to its antihistaminic effect could also play a direct inhibitory effect on eosinophil recruitment. Moreover, cetirizine was not toxic for eosinophils and did not induce degranulation, as shown by the absence of peroxidase release. Comparison between cetirizine and a PAF acether antagonist (BN 52021) suggested that cetirizine did not act by a PAF receptor-blocking activity.     

Role of purified serum components in polymorphonuclear leukocyte chemotaxis

Three highly purified serum proteins are examined for their role during leukocyte chemotaxis. In the absence of added proteins, migration of eosinophils towards eosinophil chemotactic factor and of neutrophils towards bacterial factor does not occur at all. Dose-response curves with human serum albumin (HSA) show that chemotaxis proceeds well after addition of HSA, with an optimum at 1 mg/ml. SHA has to be present in the buffer during chemotaxis since preincubation of cells followed by washing is ineffective. In contrast, alpha 2-macroglobulin and alpha 1-antitrypsin enhance leukocyte migration at doses close to physiological serum levels and affect cells irreversibly. The data suggest a complex and powerful modulating influence of serum proteins on the influx of eosinophils and neutrophils to tissue sites.     

Enhancement by a serum factor of immunoglobulin-mediated histamine release from human leukocytes.

When serum was fractionated on Sephadex G-200, the material eluted in the second and thir major peaks had a very pronounced capacity to enhance IgE- as well as IgG-mediated histamine release from the leukocytes of both normal and allergic donors. Unseparated serum on the other hand had a low capacity to stimulate anti-IgE-induced histamine release. Besides resulting in a higher histamine release, pretreatment with serum fractions also increased the rate of histamine release. Further purification revealed that the stimulating activity of the material in the second peak was mediated by a trypsin-sensitive component, probably active in low concentration.     

Inhibition of leukocyte chemotaxis by serum factor in diabetes mellitus: Selective depression of cell responses mediated by complement-derived chemoattractants

Rat neutrophil chemotactic responses to N-formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene (LT) B₄, and lipopolysaccharide-activated serum (LPS-AS) were quantitatively assessed using the micropore filter system. Cells were suspended in either normal or diabetic rat serum for testing. Diabetic donor serum did not affect migration of neutrophils in a concentration gradient of the synthetic chemotactic agents. In contrast, the migratory responses to LPS-AS were significantly less than normal in this circumstance. Summation of effects was observed when FMLP and LPS-AS, or LTB₄ and LPS-AS were simultaneously added to the test chamber, with cells suspended in normal serum. Suspended in diabetic rat serum neutrophils responded normally to the synthetic chemoattractants but the response to the activated serum was blocked. Cells previously incubated in the presence of diabetic donor serum then transferred to a culture medium for testing, presented reduced migratory responses to LPS-AS. Supramaximal, inhibitory concentrations of FMLP and LTB₄, did not influence the response of neutrophils to LPS-AS.In vivo, suppression of cellular emigration to an inflamed area was observed from the early stages of the diabetic state. The inhibitory activity of chemotaxis in diabetes mellitus was previously reported to be associated with a protein factor in plasma of the animals. It is suggested that the inhibitory factor of chemotaxis in diabetes mellitus interacts with neutrophil receptors for complement-derived chemoattractants to induce blockade of cell-oriented locomotion eitherin vitro orin vivo.     

Enhancement of human polymorphonuclear leukocyte adherence by the phospholipid mediator 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine (AGEPC).

The phospholipid mediator 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine (AGEPC) increases human polymorphonuclear leukocyte (PMN) adherence to Sephadex G-25 in manner directly proportional to the concentration of AGEPC from 2 to 650 nM. The maximal enhancement of PMN adherence of 85% +/- 23% (mean +/- SD) evoked by 650 nM AGEPC was indistinguishable in magnitude from that elicited by an optimal concentration of the chemotactic peptide N-f-Met-Leu-Phe. The results of analyses of the relative PMN adherence-enhancing activities of analogs of AGEPC suggest distinct molecular requirements for the enhancement of adherence. PMN adherence increases within 1 minute after exposure of the leukocytes to AGEPC, and the augmented adherence persists for over 30 minutes in spite of extensive washing of the leukocytes. Indomethacin partially blocked enhancement of adherence by AGEPC, suggesting that thromboxane A2 mediates in part the enhancement of adherence by AGEPC.     

Enhancement of antigen-induced leukocyte histamine release by a mononuclear cell--derived factor.

Supernatant fluids of phytohemagglutinin-stimulated mononuclear cells from ragweed-sensitive patients significantly enhanced the release of histamine from antigen-triggered leukocytes of ragweed-sensitive as well as control individuals. Supernatants of mononuclear cells from control individuals did not reveal this enhancing effect, nor was it found with the use of supernatants of unstimulated mononuclear cells of ragweed-sensitive patients or culture media with PHA alone. Supernatant fluids of phytohemagglutinin-stimulated mononuclear cells of patients sensitive to trees and grass also revealed this enhancing effect. The factor(s) responsible for the enhancement of antigen-induced histamine is heat labile and has a molecular weight of less than 10,000 daltons. The mechanism and site of action of the enhancing factor could involve initiating and/or modulating steps of the leukocyte histamine release reaction. This factor, presumably a lymphokine or a monokine, may constitute a regulating link between cell-mediated immunity and histamine-mediated hypersensitivity reactions in allergic patients.     

Endothelial protein C receptor-dependent inhibition of human eosinophil chemotaxis by protein C

BACKGROUND: Eosinophil infiltration is a characteristic feature of allergic inflammation. Allergic responses are associated with local activation of the coagulation pathway and accumulation of fibrin. OBJECTIVE: We tested whether protein C and activated protein C (APC), which are endogenous anti-inflammatory coagulation inhibitors, affect eosinophil function. METHODS: Eosinophils were from venous blood of healthy donors. Cell migration and apoptosis were studied by using micropore filter assays and fluorometry, respectively. Receptor expression was investigated by means of RT-PCR and SDS-PAGE of immunoprecipitated protein. RESULTS: Protein C and APC had no significant chemotactic effects on eosinophils. Eosinophils pretreated with protein C or APC showed significantly reduced migration toward chemoattractants. No effect of either protein C preparation was seen in eosinophil apoptosis assays. The inhibiting effect on migration was reversed by an antibody against the endothelial protein C receptor (EPCR). Synthesis of EPCR by eosinophils is suggested by demonstration of receptor mRNA expression and detection of metabolically labeled receptor protein. CONCLUSIONS: Data suggest that an EPCR is expressed by eosinophils whose activation with protein C or APC arrests directed migration. Protein C-affected eosinophil chemotaxis is a novel thrombin-independent component of the protein C pathway.     

Antigen-induced eosinophil chemotactic factor (ECF) release by human leukocytes

A complement-independent eosinophil chemotactic factor (ECF) is described which is released from peripheral leukocytes of allergic and normal human volunteers after antigen stimulation and after exposure to anti-IgE. Dose response and time-release curves for ECF and histamine run closely parallel in this system. Histamine by itself is shown to have no effect on chemotaxis at the concentrations present in antigen-induced release, but is inhibitory at very high concentrations. Evidence suggests that the ECF released from human leukocytes is derived from basophils and is similar, or identical, to the ECF released from mast cells.This work was supported by Grants 07290 and 08270 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.Publication No. 197, O'Neill Research Laboratories.Dr. Czarnetzki is recipient of the Stetler Research Fund for Women Physicians.     

Effect of rokitamycin on human polymorphonuclear leukocyte chemotaxis

The effect of rokitamycin on human polymorphonuclear leukocyte (PMNL) chemotaxis was studied in vitro and in vivo. It was found that rokitamycin in vitro at a concentration of 20 micrograms/ml caused a diminution of PMNL migration, while at lower concentrations no significant effects on migration was observed. In in-vivo studies before and after the ingestion of rokitamycin by six healthy individuals, no change on PMNL chemotaxis was found.     

Modulation of eosinophil chemotaxis by interleukin-5.

Eosinophilia and eosinophil function are regulated by cytokines such as granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5). We have investigated the modulatory role of IL-5 on N-formyl-methionyl-leucyl-phenylalanine (FMLP), neutrophil-activating factor (NAF/IL-8), platelet factor 4 (PF4), and cytokine-induced chemotaxis of eosinophils from normal individuals. These eosinophils show a small chemotactic response toward PF4 but not to NAF/IL-8 and FMLP. Preincubation of eosinophils with low concentrations of IL-5 caused significantly increased responses toward PF4 and induced a significant chemotactic response toward FMLP and NAF/IL-8. In marked contrast, IL-5 (or IL-3) priming of eosinophils from normal donors resulted in a strong inhibition of GM-CSF-induced chemotaxis. A similar decrease in the chemotactic response toward GM-CSF was observed in eosinophils derived from allergic asthmatic individuals. This finding suggests that the latter eosinophils may have had a prior exposure to IL-5 (or IL-3). Washing of the cells after priming did not abrogate the inhibition of the GM-CSF response. Our data indicate that at low concentrations IL-5 is an important modulator of eosinophil chemotaxis, causing selective upregulation or downregulation of chemotactic responses toward different agents.     

Inhibition of human eosinophil chemotaxis and of the IgE-dependent stimulation of human blood platelets by cetirizine

Cetirizine is a new anti-allergic compound with a potent, long-acting, and specific antihistaminic property. Strongly active in the therapy of urticaria and seasonal or perennial rhinitis, it has been shown to inhibit the in vivo eosinophil attraction at skin sites challenged with allergen in atopic patients. In the present work, we confirmed that, at a therapeutical concentration, this molecule had a potent inhibitory action in vitro on eosinophil chemotaxis induced either by N-formyl-Met-Leu-Phe or platelet-activating factor and also on the IgE-dependent stimulation of platelets. These observations appear in favour of a possible role for cetirizine in the modulation of inflammatory cell interactions in allergic processes.     

Subcellular localization of the eosinophil chemotactic factor (ECF) and its inactivator in human polymorphonuclear leucocytes (PMN).

An eosinophil chemotactic factor (ECF) of low MW can be released from human polymorphonuclear leucocytes (PMN) on stimulation with the Ca-ionophore, arachidonic acid and during phagocytosis. After a rapid rise of ECF activity in the supernatant a steep fall of in its activity occurred at the later times of secretion suggesting a mechanism of ECF inactivation. ECF obtained at the later times of secretion represents a stable biological activity and does not decrease on further incubation. In addition, intact PMN and ECF combined do not lead to its inactivation, while incubation of homogenized PMN with ECF decreased its activity. These data suggest the presence of an inactivator for ECF within human PMN. The purpose of the study was to localize ECF and its inactivator within human PMN. After cell disruption, differential and equilibrium gradient centrifugation, subcellular components of human PMN can be obtained which reveal eosinophilotactic (ECF) or ECF-inactivating activity. ECF activity can be recovered (in a structurally bound state) from the microsomal fraction of unstimulated and stimulated PMNs, while another portion is obtainable as a soluble, low mol. wt ECF. The PMN-derived ECF inactivator can be recovered from the peroxidase positive (azurophilic) granules and has a mol. wt of 60,000 and less. We suggest that low mol. wt ECF is derived from the plasma membrane of PMN which can be inactivated by components of the azurophilic granules. The mechanism of inactivation is still unresolved.     

Inhibition of polymorphonuclear leukocyte chemotaxis by streptokinase-streptodornase

Leukotaxis of circulating polymorphonuclear leukocytes (PMN's) appears to be a critical step in the generation of local inflammatory reactions. The present studies demonstrate a reversible, dose-dependent inhibition of PMN chemotaxis by streptokinase-streptodornase (SK-SD). This inhibition does not appear to depend upon the presence of lymphocytes or the release of chemokinetic humoral factors and phagocytic and bactericidal capacities of PMN's are unaffected by the doses of SK-SD employed.     

Assay of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase

Conditions were optimized for measuring the activity of myeloperoxidase (MPO) and the eosinophil peroxidase (EPO) with tetramethylbenzidine (TMB) as the substrate. Detergents caused a small increase in the measured activity of the purified enzymes and were required when isolated neutrophils or eosinophils were assayed. Sharp concentration optima were observed with both ionic and non-ionic detergents. Activity was also influenced by halide ions. Bromide or iodide caused up to a 7-fold increase in EPO activity and a 1.5-fold increase in MPO activity. The effect of bromide is notable because the bromide-containing detergent CETAB is often used to extract the enzymes for assay and purification. Stimulation by bromide or iodide was consistent with peroxidase-catalyzed oxidation of the halides to hypohalous acids (HOBr and HOI), which oxidized TMB. MPO catalyzes the oxidation of chloride to hypochlorus acid (HOCl), which also oxidized TMB, but chloride up to 20 mM had little effect on the assay. Both MPO and EPO catalyze thiocyanate oxidation, but the product (HOSCN) was a poor oxidant for TMB, and thiocyanate inhibited the measured activities. Stimulation by bromide or iodide could be used to facilitate detection of EPO and to distinguish between MPO and EPO. Activities could also be distinguished based on the greater sensitivity of EPO to inhibition by thiocyanate, azide, aminotriazole, and dapsone. Methods reported here may prove useful for measuring leukocyte influx into inflamed tissues, detecting MPO or EPO deficiencies, and measuring enzyme synthesis and secretion.     

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.