Accumulation of leukotriene C4 and histamine in human allergic skin reactions.

To determine whether lipoxygenase products of arachidonic acid metabolism are released in vivo during human allergic cutaneous reactions, we serially assayed chamber fluid placed over denuded skin sites for the presence of both C-6 peptide leukotrienes (e.g., LTC4, LTD4, and LTE4) and leukotriene B4 (LTB4), using radioimmune assay and HPLC separation, and compared it to histamine (assayed radioenzymatically) in 13 atopic and two nonatopic volunteers. Skin chamber sites challenged with ragweed or grass pollen antigen (250-750 protein nitrogen units/ml) for the first hour and phosphate-buffered saline (PBS) for the next 3 h were assayed hourly and compared to sites challenged with PBS alone. As assessed by HPLC, LTC4 composed greater than 85% of the C-6 peptide leukotriene released at any skin site, whereas little LTD4 or LTE4 was detected. LTC4 was present in significantly greater concentrations at antigen sites as compared to PBS-challenged sites throughout the 4-h period. Minimal concentrations of LTB4 were found throughout this time period and were not different at antigen or PBS sites. Histamine was present in significantly greater concentrations at antigen rather than PBS sites, but the pattern of release was different from that of LTC4. Peak histamine release invariably occurred during the first hour and decreased progressively thereafter, whereas the greatest amounts of LTC4 were detected during the 2nd to 4th hours. The amount of LTC4 accumulating at the site was dependent upon the dosage of antigen used in the epicutaneous challenge. We have demonstrated in this study that of the leukotrienes assessed LTC4 is released in the greatest quantity in situ during in vivo allergic cutaneous reactions and that it is present at such sites for at least 4 h after antigen challenge. Since intradermal injection of LTC4 in humans induces wheal and flare responses that persist for hours, our findings support the hypothesis that LTC4 is an important mediator of human allergic skin reactions.

Platelet activating factor increases expression of complement receptors on human neutrophils.

The phospholipid inflammatory mediator platelet activating factor (PAF) has been shown to stimulate certain functions of polymorphonuclear leukocytes (PMN). However, the effect of PAF on surface complement receptors of PMN has not been described. Using monoclonal antibodies and flow cytometry, we have assessed the effects of PAF on surface expression of membrane receptors for C3bi (CR3) and C3b (CR1) in human PMN. PAF (optimal concentration of 1 x 10(-8) M) increased CR3 190% and CR1 174% compared with unstimulated cells at 37 degrees C, while the PAF analogue lyso-PAF had no stimulatory effect. Both CR3 and CR1 responses to PAF reached maximum levels at 15-30 min. PAF effects were comparable to peak effects induced by LTB4 but less than induced by FMLP. A PAF receptor antagonist, SRI 63-441, blocked the increased complement receptor expression in a dose-dependent manner with maximal inhibition of 80-95% at 5 x 10(-6) M. Extracellular calcium had no effect on CR1 expression but slightly enhanced and EGTA partially inhibited the PAF-induced increase in CR3 expression. Simultaneous incubation with PAF and LTB4 enhanced CR3 and CR1 expression more than either agent alone. These findings indicate that PAF, alone and in combination with LTB4, can induce altered expression of complement receptors on the surface of PMN. This effect may enhance adhesion and phagocytosis by PMN at inflammatory reaction sites.

Cutaneous late phase responses--an allergic inflammatory response.

Cutaneous late phase responses have been demonstrated to occur secondary to either immune or nonimmune mast cell activation. In this article I will review studies from our laboratory that support the concept that inflammatory cells, migrating into the allergic reaction secondary to mast cell activation, are themselves activated. These activated cells release mediators that then can induce edematous, indurated cutaneous responses occurring hours after mast cell activation that have been termed "late phase cutaneous responses."

Mediator release in local heat urticaria.

We described the sixteenth reported case of local heat urticaria, in a 59-yr-old woman with erythema and angioedema upon contact with hot water or outdoor heat exposure. Immersing her hand in 39 degrees to 40 degrees C heated water resulted in an erythematous, angioedematous response sharply demarcated by the line of immersion and was associated with immediate increases in histamine concentration (18 to 135 ng/ml) and high molecular weight neutrophil chemotactic activity (two to five times prechallenge levels) in venous blood draining the challenge site. We suggest that the local heat urticarial response in this woman was a form of physical urticaria associated with release of mast cell-derived mediators, akin to cold and cholinergic urticaria.

Release of neutrophil chemotactic activity during immediate hypersensitivity reactions in humans.

Heat-stable, serum-derived chemotactic activity for neutrophils is shown in a human model of immunoglobulin E-mediated asthma. Twenty-six ragweed-sensitive subjects underwent bronchial provocation challenge using ragweed and Mecholyl. Increased neutrophil chemotactic activity was found in serum tested from 5 to 30 min after a positive ragweed-inhalation challenge, but not after negative ragweed challenge. The appearance of neutrophil chemotactic activity did not reflect the effects of bronchospasm alone, because it was not found after bronchospastic responses to Mecholyl in the same subjects. There were no accompanying changes of serum complement activity, nor evidence of inhibition of the chemotactic activity by proir exposure to antisera to the third and fifth components of complement. Ultrafiltration of serum showed chemotactic activity contained in fractions of at least 50 000 daltons. This appears to be the first demonstration of neutrophil chemotactic activity liberated during experimentally induced immunoglobulin E-mediated asthma in humans.

A prospective classification of the respiratory manifestations of pollen sensitivity.

Twenty pollen sensitive subjects were classified historically as Groups I-III based upon the elicitation of lower respiratory symptoms at (I) no time of the year, (II) only during the pollen season or (III) during and apart from the pollen season respectively. Neither antigen skin sensitivity nor antigen or mecholyl inhalational sensitivity was useful in delineating these three groups. However, pulmonary function tests during the season demonstrated changes in MMEFR and airflow at 25% vital capacity in Group II subjects, confirming our classifications.

In vivo effects of corticosteroids on human allergic responses. I. Effects of systemic administrations of steroids.

In order to better understand the effects of corticosteroids in allergic disease, the relative degrees of allergen-induced cutaneous histamine release and granulocyte accumulation were evaluated in atopic volunteers following intravenous placebo or methylprednisolone administration. In nine subjects, a single intravenous dose of methylprednisolone (1 mg/kg) did not inhibit histamine release but did significantly reduce granulocyte accumulation at both allergen and buffer-challenged sites during five and one-half to six and one-half hours following steroid injection. These findings suggest that the corticosteroid-induced anti-inflammatory effects at allergic reaction sites do not include inhibition of local mediator release. Furthermore, the steroid-induced inhibition of inflammatory cell accumulation is not specific for the allergic reaction.

Comparison of serum histamine-releasing activity and clinical manifestations in chronic idiopathic urticaria.

We found increased serum histamine-releasing activity (HRA) in 27% of patients with chronic idiopathic urticaria. Patterns of clinical manifestations were similar in those with and without serum HRA, including responses to a standard treatment regimen. In HRA+ patients the degree of serum HRA generally correlated with clinical disease activity.

Histologic responses in human skin test reactions to ragweed. IV. Effects of a single intravenous injection of steroids.

A controlled study has been carried out dealing with the early effects of a single intravenous dose of either methylprednisolone or placebo or newly developed and ongoing cellular inflammatory responses in immediate hypersensitivity skin test reactions. There was no significant difference in the tissue eosinophil responses to ragweed injected 2 hr before and 2 hr after placebo; there was a significant rise (101% +/- 39) from the second to fourth hour after antigen injection. By contrast, there was a marked decrease in the tissue eosinophil response to antigen injected 2 hr after steroids as compared to the pattern seen in the presteroid reaction. In addition, the eosinophil numbers not only did not increase from the second to fourth hour when steroids were injected at the second hour but decreased markedly. These findings suggest early suppressive effects on tissue eosinophil responses within 2 hr after steroid were administered intravenously. Also, there may be trafficking of eosinophils both into and out of these inflammatory sites during the first hours after intradermal antigen injection.

Histologic studies of human skin test responses to ragweed and compound 48/80. III. Effects of alternate-day steroid therapy.

Our previous studies have shown depressed eosinophil responses in skin test reactions to pollen antigens and compound 48/80 in those just completing a 1-wk course of daily steroids. Wheal reactions were unaffected. In this study, 6 ragweed-sensitive atopic subjects were studied before and on the seventh day ("day on") and day 8 ("day off") of a course of alternate-day steroids. Blood neutrophil levels rose on day 7 and were similar to baseline on day 8, whereas blood eosinophil levels were significantly reduced on both days 7 and 8. Neutrophil responses in skin test reactions were depressed on day 7 and normal on day 8. In contrast, the tissue eosinophil responses were depressed significantly, and to similar degree, on both days 7 and 8. These findings are of potential significance in evaluating the clinical effects of steroids in allergic diseases.

Factors in the tissue eosinophil responses in human immediate hypersensitivity reactions.

Factors underlying the eosinophil responses in the tissue sites of immediate hypersensitivity reactions have been investigated. A quantitative assessment of the number of eosinophils appearing in skin test reactions to ragweed antigen and compound 48/80 in ragweed-sensitive humans has been compared with several other parameters. There was a moderate, statistically significant, correlation with serum levels of IgE-class antiragweed antibody; also, eosinophil responses were minimal or absent in minimally positive threshold dilution skin tests. Tissue eosinophil responses were generally limited to those with baseline blood eosinophil levels of at least 150 mm3; however, there was no correlation between blood and tissue eosinophil levels in individual subjects. These findings suggest that the pathogenesis of the observed tissue eosinophil responses may be multifactorial.

Antigen-induced neutrophil chemotactic activity in man. Correlation with bronchospasm and inhibition by disodium cromoglycate.

We have previously reported increased neutrophil chemotactic activity in sera obtained after positive antigen inhalation responses in atopic subjects. This report describes the kinetics of appearance of this serum activity and the effects of antigen dose and disodium cromoglycate pretreatment on the response in 10 ragweed-sensitive subjects. Significantly increased chemotactic activity was present as early as 1 min, peaked at 10 min, and persisted through 24 hr after inhalation of antigen. The increased chemotactic activity correlated with the degree of bronchospasm induced by antigen inhalation and the amount of antigen administered. The increased chemotactic activity and bronchospasm were blocked by administration of disodium cromoglycate prior to antigen challenge. These findings are consistent with a postulated antigen-induced anaphylactic release of chemotactic activity. The correlation of this activity with the degree of bronchospasm and its appearance after administration of even small doses of antigen suggest that this activity may be important in antigen-mediated bronchospasm.

Quantitation by myeloperoxidase assay of neutrophil accumulation at the site of in vivo allergic reactions.

Skin window techniques to investigate neutrophil inflammatory reactions in human skin have been limited by cellular distortion and difficulties in quantitation. We have developed a quantitative approach based on the assessment of the myeloperoxidase (MPO) released from sonicated membrane filters to which exuding inflammative cells had adhered. Our studies have shown that (1) a standard curve can be derived for each subject from the linear relationship between amounts of MPO released and designated numbers of blood neutrophils which have been aspirated onto such filters; (2) the number of neutrophils adhering to filters appended to skin window sites can then be reliably estimated by comparing the amount of MPO released by sonication of such filters with the standard curve; and (3) neutrophil accumulation is greater at pollen-challenged than control sites in sensitive subjects.

Plasma concentrations of histamine measured by radioenzymatic assay: effects of histaminase incubations.

To help resolve the current uncertainty as to whether assays for plasma histamine are measuring non-histamine compounds as well, we compared the effects of prior incubation with histaminase and buffer on measurements in (1) normal plasma, (2) buffer and normal plasma to which several amounts of exogenous histamine had been added, and (3) plasma obtained after inhalation-induced asthma or form the site of a local heat urticaria challenge. As measured by the radioenzymatic technique, low (1 to 4 ng/ml) levels of histamine-like material were present in normal plasma after incubation with either histaminase or buffer. In contrast, histaminase (but not buffer) incubation markedly reduced measured histamine in all other specimens. Exogenous histamine in buffer was reduced almost 100% by histaminase, whereas the degree of reduction in plasma specimens varied directly with the starting histamine level. Therefore it appears that low levels of histaminase-resistant material reacting in histamine assays is present in normal plasma. The use of histaminase incubation appears to be helpful in differentiating this from true histamine release in allergic reactions.

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.

Further characterization and biologic activity of ragweed antigen--induced neutrophil chemotactic activity in man.

We have previously described the appearance in serum of increased neutrophil chemotactic activity (NCA) during bronchospasm induced by inhalation of ragweed antigen in ragweed-sensitive subjects. This NCA is non-complement derived, appears within 1 min after antigen inhalation, and is not seen after methacholine-induced bronchospasm. This article describes further characterization of this chemotactic activity and correlation with in vivo leukocytosis. NCA consistently eluted in the void volume (fraction I) after Sephadex G-150 chromatography of patient serum obtained 10 min postchallenge. Fraction I contained 94% of the NCA of postchallenge whole serum. Both postchallenge whole serum and fraction I deactivated neutrophils to autologous chemoattractants and complement-derived chemotactic factors, but not serum-independent chemotactic factors. NCA was chemotactic for neither human nor guinea pig eosinophils, nor for human mononuclear cells. A significant increase of circulating neutrophils was seen only after antigen-induced bronchospasm and correlated with the increase in NCA. Thus, NCA represents another inflammatory mediator of probable mast cell origin that may explain, at least partially, the accumulation of neutrophils observed in the peripheral blood, skin, and bronchial wall after immediate hypersensitivity reactions.

Antigen-induced local mediator release and cellular inflammatory responses in atopic subjects.

We report comparisons of histamine release and neutrophil exudation in skin-window sites of 27 pollen-sensitive subjects challenged with antigen or buffer. More histamine was released within 30 min into appended collection chambers in antigen sites vs control sites. There were also more neutrophils adhering to membrane filters applied for 1 hr to the blister bases in antigen-challenged sites vs buffer sites. Comparison of skin-test extinction dilution titer, histamine release, and neutrophil accumulation in antigen-challenged sites in individual subjects showed that (1) there was no correlation between degrees of local histamine and neutrophil accumulation, (2) the increase in histamine but not in neutrophils correlated inversely with the concentration of antigen required to elicit a minimum wheal, and (3) both histamine and neutrophil increases were induced by antigen in a dose-dependent manner.

Modulation of serum histamine releasing activity in chronic idiopathic urticaria.

BACKGROUND: Sera of about 30% of patients with chronic idiopathic urticaria (CIU) have increased histamine releasing activity (HRA+) on normal basophils. It is not known whether other CIU sera would be HRA+ if a more sensitive histamine release assay was used. Although most HRA+ CIU sera appear to have anti Fc(epsilon)R1 activity, it is not known whether post-binding basophil intracellular events are similar to those after anti-IgE stimulation. RESULTS: In the presence of D2O, the HR stimulated by 28 previously documented HRA- sera increased from 4+/-0.4 to 21+/-4% with 13 of the 28 sera now considered HRA+. No previous HRA sera was HRA+ with IL-3 treated cells. Histamine release induced by both HRA+ sera and anti-IgE were inhibited by genistein, and Ca2+/Mg2+ depletion but not by bisindoylmaleimide. HRA+ sera induced prominent HR from normal basophils with little surface IgE, but induced no increased HR from basophils unresponsive to anti-IgE. CONCLUSIONS: Up to 61% of CIU sera will induce increased HR from normal basophils in a sufficiently sensitive assay system. HR induced by most HRA+ sera is more prominent in basophils with very little surface IgE. However, there may be similar post-binding intracellular activation pathways following stimulation by HRA+ sera and anti-IgE.

Comparison of plasma histamine and cyclic nucleotides after antigen and methacholine inhalation in man.

Serial determinations of plasma histamine and cyclic nucleotides (adenosine monophosphate [AMP] and guanosine monophosphate [GMP]) were performed after inhalation of antigen and methacholine in four groups of subjects. In the first group, consisting of six antigen-sensitive subjects exhibiting bronchospasm after inhalation of ragweed or grass antigen, plasma histamine was elevated within 2 min and persisted for 30 min after inhalation of antigen. Peak histamine levels were between 18 to 80 ng/ml. In the second group, consisting of four nonatopic subjects, neither bronchospasm nor histamine was observed, despite inhalation of the same or 10-fold increased concentrations of antigen. In the third group, consisting of six subjects (three atopic and three nonatopic) exhibiting bronchospasm after inhalation of 2.5 to 10 mg of methacholine, sustained increases of histamine began at 1 min and persisted for 60 min after inhalation of methacholine. In the fourth group, seven subjects (two atopic, five nonatopic) without demonstrable bronchospasm despite inhalation of 2.5- to 10-fold increased doses of methacholine, no histamine was detected in the plasma at any time after inhalation of methacholine. Serial measurements of cyclic nucleotides showed no consistent changes in serum levels of cyclic AMP or cyclic GMP following inhalation challenge. We conclude that serum levels of histamine but not cyclic nucleotides change during bronchospasm induced by either antigen or methacholine.