The early history of the eosinophil.

In 1879 Paul Ehrlich published his technique for staining blood films and his method for differential blood cell counting using coal tar dyes and mentions the eosinophil for the first time. Eosin is a bright red synthetic dye produced by the action of bromine on fluorescein and stains basic proteins due to its acidic nature. It was discovered in 1874 by Heinrich Caro, Director of the German chemical company Badische Anilin- und Soda-Fabrik. Ehrlich introduced the term 'eosinophil' to describe cells with granules (which he called alpha-granules) having an affinity for eosin and other acid dyes. He also observed black-staining, indulinophilic, beta-granules in bone marrow-derived eosinophils, which were probably immature crystalloid granules in eosinophil myelocytes. Ehrlich described the features of the alpha-granule and the cell's distribution in various species and tissues. He speculated correctly that the alpha-granule contents were secretory products and described several causes of eosinophilia including asthma, various skin diseases, helminths and reactions to medications. However, the cell was almost certainly observed by others before Ehrlich. In 1846 Thomas Wharton Jones (1808-1891) described 'granule blood cells' in the lamprey, frog, fowl, horse, elephant and man. He 'borrowed' the term granule cell from Julius Vogel (1814-1880) who had observed similar cells in inflammatory exudates. Vogel in turn was aware of the work of the Gottlieb (Théophile) Gluge (1812-1898) who used the term 'compound inflammatory globules' to describe cells in pus and serum. Almost 20 years before Ehrlich developed his staining methods, Max Johann Sigismund Schultze (1825-1874) performed functional experiments on coarse granular cells using a warm stage microscopic technique and showed they had amoeboid movement and phagocytic abilities. Although these early investigators recognised distinct granular cells Ehrlich's use of stains was a landmark contribution, which heralded modern studies on eosinophils and other blood leucocytes.

Elevations in T-helper-2-initiating cytokines (interleukin-33, interleukin-25 and thymic stromal lymphopoietin) in lesional skin from chronic spontaneous ('idiopathic') urticaria.

BACKGROUND: The mechanism of wealing in chronic spontaneous urticaria (CSU) is largely unknown. We previously demonstrated increased expression of T-helper 2 [interleukin (IL)-4 and IL-5] cytokines in skin biopsies from CSU. This suggested that Th2-initiating cytokines [IL-33, IL-25 and thymic stromal lymphopoietin (TSLP)], released through innate immune mechanisms, may play a role in pathogenesis. OBJECTIVES: To identify Th2-initiating cytokines in lesional and nonlesional skin from patients with CSU and to compare the results with a control group. METHODS: Paired biopsies (one from a 4-8 h spontaneous weal and one from uninvolved skin) were taken from eight patients with CSU and nine control subjects, and studied by immunohistochemistry and confocal microscopy. RESULTS: There were increases in IL-4(+) and IL-5(+) cells in lesional skin vs. controls (P = 0·03 and P < 0·001, respectively) and marked elevations in the numbers of IL-33(+), IL-25(+) and TSLP(+) cells in the dermis of lesional skin vs. both nonlesional skin (P = 0·002, P = 0·01 and P = 0·04, respectively) and controls (P = 0·001, P < 0·001 and P = 0·005, respectively). There was also a correlation between the numbers of IL-33(+) and IL-25(+) cells (r = 0·808, P = 0·015). IL-33 localized to CD31(+) endothelial cells, CD90(+) fibroblasts, CD68(+) macrophages and tryptase(+) mast cells, whereas IL-25 was expressed by epithelial cells, mast cells and major basic protein-positive eosinophils. IL-33 and IL-25 were constitutively expressed in the epidermis of both controls and patients with CSU. CONCLUSIONS: Increased expression of Th2-initiating cytokines in lesional skin in CSU suggests that innate pathways might play a role in the mechanism of wealing. As Th2-initiating cytokines play a role in mast cell activation, inflammation and vascular leakage in CSU, these findings may also have therapeutic implications.

Calcitonin gene-related peptide and vascular endothelial growth factor are expressed in lesional but not uninvolved skin in chronic spontaneous urticaria.

BACKGROUND: The mechanisms for producing weals in chronic spontaneous (idiopathic) urticaria (CSU) are incompletely understood. Leucocyte infiltration with vascular leakage and expression of the potent vasoactive agents' calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor (VEGF) are features of late-phase allergic skin reactions, previously proposed as a model of CSU. OBJECTIVE: To measure CGRP and VEGF expression in lesional and non-lesional skin from CSU patients and to compare results with a control group. METHODS: Eight paired biopsies (one from 4-8 h spontaneous weals and one from uninvolved skin) were taken from eight patients with CSU and nine control subjects and studied by immunohistochemistry and confocal microscopy. RESULTS: Lesional skin in CSU contained significantly more CGRP+ and VEGF+ cells than non-lesional skin. No significant differences were observed in CGRP and VEGF expression between non-lesional skin and controls. In lesional skin, VEGF and CGRP co-localised to UEA-1+ blood vessels. CGRP was also expressed by neutrophils and eosinophils and to a lesser extent by CD90(+) fibroblasts, mast cells, CD3(+) and CD68(+) cells. CGRP and VEGF expression was not related to the duration of disease. CONCLUSION AND CLINICAL RELEVANCE: Increased expression of CGRP and VEGF in lesional, but not uninvolved, skin indicates that these potent vasoactive agents may play a role in wealing and tissue oedema in CSU so representing novel targets in therapy.

Elevations in vascular markers and eosinophils in chronic spontaneous urticarial weals with low-level persistence in uninvolved skin.

BACKGROUND: In chronic spontaneous urticaria (CSU) mast cell activation together with inflammatory changes in the skin are well documented and may play an important role in mechanisms of tissue oedema. OBJECTIVES: To confirm and extend these observations by measuring microvascular markers, leucocytes and mast cell numbers in lesional and uninvolved skin and to compare findings with a control group. METHODS: Paired biopsies (one from 4-8-h spontaneous weals and one from uninvolved skin) were taken from eight patients with CSU and nine control subjects and studied using immunohistochemistry and confocal microscopy using the lectin Ulex europaeus agglutinin 1 (UEA-1). RESULTS: Lesional skin in CSU contained significantly more CD31+ endothelial cells; CD31+ blood vessels, neutrophils, eosinophils, basophils and macrophages; and CD3+ T cells than nonlesional skin. Increased vascularity was confirmed by confocal imaging using the lectin UEA-1. Uninvolved skin from CSU contained significantly more CD31+ endothelial cells, CD31+ blood vessels and eosinophils compared with the control subjects. There was a threefold increase in mast cell numbers when CSU was compared with controls but no difference was observed between lesional and uninvolved skin. CONCLUSIONS: Increased vascular markers together with eosinophil and neutrophil infiltration are features of lesional skin in CSU and might contribute to tissue oedema. Eosinophils and microvascular changes persist in uninvolved skin, which, together with increased mast cells, suggests that nonlesional skin is primed for further wealing.

Immunotherapy is allergen-specific: a double-blind trial of mite or timothy extract in mite and grass dual-allergic patients.

BACKGROUND: One hundred years ago, Noon [Lancet 1911;1:1572-1573], using conjunctival provocation testing (CPT), was the first to demonstrate the effectiveness of subcutaneous immunotherapy (SCIT) in grass-allergic subjects with hay fever. In this centenary year, we present data that, by use of CPT and allergen-specific IgG, replicate this observation and additionally confirm the allergen specificity of SCIT by using a double-blind design employing either grass or mite SCIT in dual grass- and mite-allergic individuals. METHODS: Twenty adults (11 females) with perennial rhinoconjunctivitis and exacerbation of symptoms during the grass pollen season and in the autumn had immediate skin and conjunctival sensitivity and raised specific IgE to both Dermatophagoides farinae and Phleum pratense. Participants were randomly assigned to either timothy or D. farinae immunotherapy for 3 years. CPT and specific IgG tests to both allergens were performed annually. After 3 years, subjects gave their blinded overall evaluation. RESULTS: Six mild-to-moderate general reactions occurred in 2 timothy- and 4 mite-treated patients. Four of these patients and 2 other patients withdrew from the study. Seven patients in each group completed the study. After 3 years of immunotherapy, the timothy CPT threshold concentration had increased 16- fold in timothy-treated patients (p < 0.05; between-group change, p < 0.05). The increase in the mite CPT threshold in mite- compared to grass-treated patients was 31-fold (p < 0.05). The overall assessment of conjunctival sensitivity was highly significant in favour of treatment (p < 0.015), as was that of allergen-specific IgG (p < 0.0001). CONCLUSIONS: Allergen immunotherapy is allergen species-specific, as judged by decreased conjunctival sensitivity and changes in allergen-specific IgG concentrations.

Immunoglobulin E-independent major histocompatibility complex-restricted T cell peptide epitope-induced late asthmatic reactions.

Intradermal administration of short overlapping peptides derived from chain 1 of the cat allergen Fel d 1 (FC1P) that did not cross-link IgE, elicited isolated late asthmatic reactions with no visible early or late cutaneous response in 9/40 cat-allergic asthmatics. Four of the nine were human histocompatibility leukocyte antigen DR13-positive, as compared with only 1/31 nonreactors. The other five reactors expressed either DR1 or DR4. To confirm major histocompatibility complex restriction, fibroblast cell lines transfected with HLA-DR molecules were used to present FC1Ps to cat allergen-specific T cell lines derived from subjects before peptide injection. FC1P3 (peptide 28-44 of Fel d 1 chain 1) was recognized in the context of DR13 alleles (DRB1*1301, 1302) and induced specific T cell proliferation and IL-5 production. T cells from a DR1(+) responder proliferated and produced IL-5 in the presence of FC1P3 and DR1 (DRB1*0101) fibroblast cell lines, whereas T cells from a DR4(+) subject recognized FC1P2 (peptide 22-37) when presented by DRB1*0405. We conclude that short, allergen-derived peptides can directly initiate a major histocompatibility complex-restricted, T cell-dependent late asthmatic reaction, without the requirement for an early IgE/mast cell-dependent response, in sensitized asthmatic subjects.

An eosinophil leukocyte chemotactic factor of anaphylaxis.

The capacity of actively or passively sensitized guinea pig lung to react with antigen to release a factor specifically chemotactic for eosinophil leukocytes (ECF-A) has been demonstrated. The release of ECF-A was also accompanied by the elaboration of both histamine and SRS-A and the appearance of all these mediators exhibited a similar response in terms of the time course of passve sensitization, the effect of antigen dose, the time course of release, divalent cation dependence and enhancement by the presence of succinate or maleate. Decomplementation by the administration of purified cobra venom factor had no effect on the antigen-induced release of ECF-A from actively or passively sensitized lung fragments. When fragments of guinea pig lung were passively sensitized with fractions of guinea pig 7S IgG, only the IgG(1)-containing fractions prepared tissue for the antigen-induced release of ECF-A. Histamine, SRS-A, bradykinin, serotonin, and the prostaglandins PGE(1), PGE(2), and PGF(2alpha) were not eosinophilotactic per se; neither was ECF-A detected following the incubation of these agents with sensitized lung in the absence of antigen. Both eosinophilotactic activity and SRS-A survived extraction in 80% ethanol and evaporation to dryness. SRS-A, however, withstood boiling in alkaline solution for 20 min, whereas ECF-A activity was abolished by this procedure. SRS-A and ECF-A could also be separated by gel filtration. ECF-A activity was completely recovered following its passage through a column of Sephadex G-25 and had an estimated molecular weight of between 500 and 1000. On the basis of size and a formation mechanism independent of the complement system, ECF-A is distinguishable from a previously described complement-dependent eosinophilotactic factor (ECF-C). Thus, ECF-A represents a hitherto undescribed agent which selectively attracts eosinophil leukocytes.

A prealbumin activator of prekallikrein. 3. Appearance of chemotactic activity for human neutrophils by the conversion of human prekallikrein to kallikrein.

Human plasma kallikrein has been shown to directly and selectively attract human neutrophils from a mixed leukocyte population. The capacity of plasma kallikrein to be chemotactic and to generate the nonapeptide bradykinin was maintained during progressive purification. While neither highly purified prekallikrein nor the prealbumin Hageman factor fragments were chemotactic alone, their interaction so as to convert prekallikrein to kallikrein yielded both chemotactic and kinin-generating activity. Both functions of kallikrein were inhibited by treatment with diisopropyl fluorophosphate, indicating an essential role for the active site of the enzyme in the expression of its chemotactic activity.

Intracellular application of guanosine-5'-O-(3-thiotriphosphate) induces exocytotic granule fusion in guinea pig eosinophils.

The mechanism of eosinophil secretion was studied in guinea pig eosinophils by measuring release of hexosaminidase from cell suspensions (greater than 98% pure) permeabilized with streptolysin-O and by whole-cell patch-clamp capacitance measurements. It is shown that release of eosinophil granule components occurs by an exocytotic mechanism in which individual granules fuse with the plasma membrane. Exocytosis can be induced by intracellular application of the nonhydrolyzable GTP analog guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), suggesting the involvement of a GTP-binding protein. The activation is modulated by the intracellular calcium concentration, with activation by GTP-gamma-S inducing transient elevations in the concentration of Ca2+. Thus, the nature and regulation of the release mechanism appear to be very similar to that of the mast cell and neutrophil.

The kinetics of allergen-induced transcription of messenger RNA for monocyte chemotactic protein-3 and RANTES in the skin of human atopic subjects: relationship to eosinophil, T cell, and macrophage recruitment.

The C-C chemokines RANTES and monocyte chemotactic protein-3 (MCP-3) are potent chemoattractants in vitro for eosinophils and other cell types associated with allergic reactions. We tested the hypothesis that the allergen-induced infiltration of eosinophils, T cells, and macrophages in the skin of atopic subjects is accompanied by the appearance of mRNA+ cells for RANTES and MCP-3. Cryostat sections were obtained from skin biopsies from six subjects 6, 24, and 48 h after allergen challenge. Tissue was processed for immunocytochemistry (ICC) and for in situ hybridization using 35S-labeled riboprobes for RANTES and MCP-3. In contrast to diluent controls, allergen provoked a significant increase in mRNA+ cells for MCP-3, which peaked at 6 h and progressively declined at 24 and 48 h. This paralleled the kinetics of total (major basic protein positive [MBP]+) and activated (cleaved form of eosinophil cationic protein [EG2]+) eosinophil infiltration. The allergen-induced expression of cells mRNA+ for RANTES was also clearly demonstrable at 6 h. However, the numbers were maximal at 24 h and declined slightly at the 48-h time point. The number of mRNA+ cells for RANTES paralleled the kinetics of infiltration of CD3+, CD4+, and CD8+ T cells whereas the number of CD68+ macrophages was still increasing at 48 h. These data support the view that MCP-3 is involved in the regulation of the early eosinophil response to specific allergen, whereas RANTES may have more relevance to the later accumulation of T cells and macrophages.

Adhesion to fibronectin prolongs eosinophil survival.

We have investigated the effect of adhesion to fibronectin (Fn) on the survival of eosinophils in culture. Peripheral blood eosinophils from normal human donors were separated by immunomagnetic selection and cultured in RPMI on Fn- (100 micrograms/ml) coated microtiter plates for up to 96 h. Survival was measured by trypan blue exclusion. There was a significant enhancement of eosinophil survival with Fn as compared with both bovine serum albumin-coated and uncoated wells (p < 0.05-0.01). Fn-induced eosinophil survival was comparable to that obtained with exogenous interleukin 3 (IL-3) or granulocyte/macrophage colony-stimulating factor (GM-CSF) and was inhibitable by antibodies against Fn, very late antigen 4 (VLA-4), IL-3, and GM-CSF. Supernatants from Fn-, but not BSA-coated wells contained picogram amounts of IL-3 and GM-CSF, and eosinophils cultured on Fn for 24 h expressed mRNA for GM-CSF as determined by in situ hybridization. Therefore, Fn prolongs eosinophil survival in culture by triggering autocrine generation of cytokines by eosinophils. Since neutrophils lack VLA-4, this could provide a partial explanation for the preferential accumulation of eosinophils at sites of allergic inflammation, as well as the predominant tissue localization of eosinophils in healthy individuals.

Expression of mRNA and immunoreactivity for the granulocyte/macrophage colony-stimulating factor in activated human eosinophils.

Using in situ hybridization, we have shown that activated human peripheral blood eosinophils express mRNA for granulocyte/macrophage colony-stimulating factor (GM-CSF). Between 15 and 27% of eosinophils gave positive hybridization signals for GM-CSF mRNA after stimulation with the calcium ionophore A23187 or interferon gamma, and 4 and 6% after incubation with interleukin 3 (IL-3) or IL-5. Activated eosinophils also gave specific immunoreactivity with an anti-GM-CSF polyclonal antibody, suggesting translation of the mRNA. These data indicate that eosinophils may be an important source of GM-CSF at sites of allergic inflammation. Furthermore, the identification of GM-CSF production by human eosinophils suggests that the pro-inflammatory potential of this cell type may be substantially greater than hitherto recognized.

Messenger RNA expression of the cytokine gene cluster, interleukin 3 (IL-3), IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor, in allergen-induced late-phase cutaneous reactions in atopic subjects.

Cryostat sections from skin biopsies from 24-h allergen-induced late-phase cutaneous reactions (LPR) in 14 human atopic subjects were hybridized with 35S-labeled RNA probes for a number of cytokines. mRNA was detected for interleukin 3 (IL-3) (8/14), IL-4 (10/14), IL-5 (11/14), and granulocyte/macrophage colony-stimulating factor (GM-CSF) (13/14). Only 5 of 14 gave hybridization signals for IL-2, and 0 of 14 for interferon gamma. Biopsies from diluent controls gave only occasional weak signals. These results suggest that cells infiltrating the site of the 24-h LPR transcribe mRNA for the IL-3, IL-4, IL-5, and GM-CSF gene cluster and support the hypothesis that atopy is associated with preferential activation of cells having a similar cytokine profile to the murine T helper type 2 subset.

Dysregulation of interleukin 4, interleukin 5, and interferon gamma gene expression in steroid-resistant asthma.

In steroid-resistant (SR) asthma, there is a lack of clinical responsiveness to oral prednisone. Previous studies indicate that this may be explained by the effect of the combination of interleukin 2 (IL-2) and IL-4 on glucocorticoid receptor binding affinity. By contrast, steroid-sensitive (SS) asthmatics respond well to glucocorticoids, and this is accompanied by a decrease in the numbers of bronchoalveolar lavage (BAL) messenger RNA+ (mRNA+) cells expressing IL-4 and IL-5, and an increase in interferon gamma (IFN-gamma) transcripts. In the present study, we hypothesized that SR asthma is associated with alterations in T helper types 1/2 (Th2/Th1)-type cytokine gene expression. BAL was performed in six SR asthmatics and six SS asthmatics, before and after a 1-wk course of 40 mg daily prednisone. mRNA+ cells for IL-2, IL-4, IL-5, and IFN-gamma was measured by in situ hybridization using 35S-labeled RNA probes. Before prednisone therapy, there were significantly greater numbers of BAL cells (per 1,000) expressing IL-2 mRNA (p < 0.01) and IL-4 mRNA (p < 0.05) in SR asthmatics as compared with SS asthmatics, but no differences between the two groups in the numbers of BAL cells expressing IFN-gamma or IL-5 mRNA expression were observed. After a 1-wk course of prednisone, IL-2 expression was not altered in either group. However, SS asthmatics had a significant decrease in the numbers of BAL cells expressing mRNA for IL-4 (p < 0.01) and IL-5 (p < 0.001), and a rise in the numbers of IFN-gamma mRNA+ cells (p < 0.01). In contrast, after prednisone treatment, SR asthmatics had no significant change in either the number of BAL cells expressing mRNA for IL-4 or IL-5. Of note, there was an unexpected decrease in the numbers of IFN-gamma mRNA+ cells (p = 0.05). Our current findings indicate that SR asthma is associated with a dysregulation of the expression of the genes encoding for Th2/Th1 cytokines in airway cells and is compatible with the concept that a combination of IL-2 and IL-4 induce glucocorticoid (GR) binding affinity and T cell responsiveness to glucocorticoids.

Alcohol consumption is not protective for systemic lupus erythematosus.

OBJECTIVE: Several studies have suggested that alcohol drinking is protective for the development and progression of systemic lupus erythematosus (SLE). However, a protopathic bias might also explain this apparent association. Our objective was to investigate the association between alcohol consumption and incidence of SLE in a data set that has information on both current and pre-diagnostic alcohol consumption. METHODS: We performed an Internet-based case-control study of SLE. Cases were diagnosed within 5 years of the study and met > or =4 American College of Rheumatology criteria for SLE. The control participants were tightly matched to cases on demographic and socio-economic characteristics using a propensity score. Participants completed an online exposure assessment. We used conditional logistic regression analyses to test the association of current and pre-diagnostic alcohol consumption with SLE. RESULTS: The sample comprised 114 cases with SLE and 228 matched controls. Current drinking (>2 days per week) was inversely associated with SLE (OR 0.35, 95% CI 0.13 to 0.98). Having more than two drinks per day was also inversely associated with SLE (OR 0.41, 95% CI 0.18 to 0.93). However, alcohol consumption before SLE diagnosis was not associated with the risk of SLE (p> or =0.4). Analysis of the change in drinking habits showed that people with lupus were more likely to quit drinking before (OR 2.25, 95% CI 0.96 to 5.28) or after (OR 2.38, 95% CI 0.88 to 6.49) being given the SLE diagnosis. CONCLUSIONS: Our results show that alcohol consumption before SLE diagnosis is not associated with the risk for SLE, and that individuals who develop SLE are more likely to quit.

Early production of thymic stromal lymphopoietin precedes infiltration of dendritic cells expressing its receptor in allergen-induced late phase cutaneous responses in atopic subjects.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is an interleukin (IL)-7-like cytokine that triggers dendritic cell-mediated T helper (Th)2 inflammatory responses through a receptor consisting of a heterodimer of the IL-7 receptor alpha (IL-7Ralpha) chain and the TSLP receptor (TSLPR), which resembles the cytokine receptor common gamma chain. Dendritic cells activated by TSLP prime development of CD4(+) T cells into Th2 cells contributing to the pathogenesis of allergic inflammation. We hypothesized that allergen exposure induces expression of TSLP and results in recruitment of TSLPR bearing cells in the cutaneous allergen-induced late-phase reaction (LPR) in atopic subjects. METHODS: Skin biopsies were obtained from atopic subjects (n = 9) at various times after cutaneous allergen challenge. In situ hybridization and immunohistochemistry were used to determine TSLP mRNA expression and to measure infiltration of TSLPR(+) DC in skin LPR. RT-PCR and flow cytometry were employed to analyse TSLPR expression on isolated blood DC. RESULTS: Allergen-induced skin TSLP expression occurred as early as 1 h after allergen challenge, whereas TSLPR(+) and CD11c(+) cells infiltrated relatively late (24-48 h). The majority of TSLPR(+) cells were DC co-expressing blood DC antigen-1 (BDCA-1) or BDCA-2. Freshly isolated blood DC expressed both TSLPR and IL-7Ralpha chains. Maturation and stimulation with TSLP or polyriboinosinic-polyribocytidylic acid in vitro upregulated the expression of both TSLPR and IL-7Ralpha chains in DC but not in chemoattractant receptor-homologous molecule expressed on Th2 cells(+) CD4(+) T cells. CONCLUSION: The data suggest that TSLP plays a role in augmenting, through DC recruitment and activation, the development of Th2-type T cells in allergic inflammation.

Skin reactivity and local cell recruitment in human atopic and nonatopic subjects by CCL2/MCP-1 and CCL3/MIP-1alpha.

BACKGROUND: Monocyte chemotactic protein (MCP-1/CCL2), the ligand for CCR2 and CCR5, and macrophage inflammatory protein-1alpha (MIP-1alpha/CCL3), the ligand for CCR1 and CCR5, are potent chemo-attractants in vitro and produce lesions in experimental animals, which resemble immediate and delayed-type hypersensitivity (DTH) reactions. CCL3 induces mononuclear cell and granulocyte infiltration in human atopic and nonatopic skin. Whether CCL2 (MCP-1) has comparable activity in man is uncertain as is the capacity of both the chemokines to elicit immediate- and DTH-like reactions in humans. METHODS: Inflammatory cells were counted by immunohistochemistry in 24 and 48-h skin biopsies from atopics and nonatopics after intradermal injection of CCL2 and CCL3. Immediate (15 min) wheals-and-flares and delayed (24 and 48 h) indurations were also recorded. RESULTS: Both chemokines induced immediate- (15 min) and delayed (24 and 48 h) reactions, which were associated with significant infiltrations of CD68+ macrophages, CD3+, CD4+ (but not CD8+) T cells, neutrophils, and eosinophils in biopsies from injection sites. CCL2, but not CCL3, also induced infiltration of basophils. Neither chemokine produced significant changes in the numbers of tryptase+ cutaneous mast cells. There were no differences in the pattern of skin reactivity or the numbers of infiltrating leukocytes in response to CCL2 and CCL3 between atopic and nonatopic subjects. In general, maximal infiltration of inflammatory cells was observed at the 24-h, rather than the 48-h, time point. CONCLUSIONS: CCL2 and CCL3 induce both immediate and delayed skin reactions in atopics and nonatopics, and evoke a similar profile of local T cell/macrophage and granulocyte recruitment which, in general, confirm previous in vitro findings and in vivo experimental animal data.

Identification and partial characterization of an exercise-induced neutrophil chemotactic factor in bronchial asthma.

A heat-stable neutrophil chemotactic factor (NCF) has been identified in the serum of 13 atopic asthmatic subjects after treadmill exercise. Peak activity was detected at 10 min and returned to prechallenge values by 1 h. No NCF activity was detected in the sera of three nonasthmatic atopic or four normal nonatopic individuals performing the same task. NCF produced by exercise (NCFEX) had a similar time-course of release to NCF provoked by specific antigen (NCFAG). The appearance of circulating NCFEX and NCFAG closely paralleled the fall in peak expiratory flow rate/forced expiratory volume in 1 s (PEFR/FEV1). Histamine challenge in atopic asthmatics at concentrations giving a comparable change in PEFR/FEV1 to that evoked by exercise or inhaled antigen was not associated with the appearance of circulating NCF. In seven subjects NCFEX release was inhibited by prior administration of disodium cromoglycate. NCFEX and NCFAG eluted as single peaks of activity when applied separately to columns of Sephadex G-200, and both were an estimated 750,000 daltons. NCFEX and NCFAG also eluted as single peaks of activity, at between 0.15 and 0.30 M NaCl, following anion exchange chromatography on DEAE-Sephacel (pH 7.8). The isoelectric points of NCFEX and NCFAG were virtually identical (between pH 6.0 and 6.5) as determined by chromatofocusing on Polybuffer Exchanger 94. The activities of NCFEX and NCFAG were substantially reduced, in both a time- and dose-dependent fashion, after incubation with trypsin and chymotrypsin. Partially purified NCFEX and NCFAG promoted both stimulated random migration (chemokinesis) as well as directional migration (chemotaxis). These experiments indicate that NCFEX and NCFAG might be identical substances and raise the possibility that mediators by hypersensitivity are released during exercise-induced asthma in atopic subjects.

Platelet-activating factor. A potent chemotactic and chemokinetic factor for human eosinophils.

Platelet-activating factor (PAF-acether), an inflammatory mediator with a wide range of biological activities including neutrophil aggregation and chemotaxis, was studied for its effect on human eosinophil locomotion (chemotaxis and chemokinesis). Human eosinophils (25-95% purity) were obtained from donors with a variety of diseases associated with hypereosinophilia. PAF-acether elicited directional locomotion of eosinophils, in a time- and dose-dependent fashion, at concentrations from 10(-5) to 10(-8) M; lyso-PAF had minimal activity over the same dose range. Compared with PAF-acether, the eosinophil locomotory responsiveness of leukotriene B4 (LTB4), histamine, and the valyl- and alanyl-eosinophil chemotactic factor of anaphylaxis (ECF-A) tetrapeptides was negligible. Conversely, neutrophil responsiveness to PAF-acether (optimum 10(-6) M) was comparable in effect to LTB4 (optimum dose 10(-8) M). It was shown that PAF-acether elicited both chemotaxis and chemokinesis of eosinophils. Comparison of normal density and light density eosinophils revealed no qualitative difference in the response to PAF-acether and the other chemoattractants, although the light density cells seemed to demonstrate a greater degree of locomotion to PAF-acether and LTB4. Thus, PAF-acether appears to be a potent eosinophilotactic agent which may play a role in inflammatory reactions characterized by eosinophil infiltration.

Human granulocyte/pollen-binding protein. Recognition and identification as transferrin.

Normal human serum was found to contain a heat-stable protein which promoted the binding of granulocytes to timothy grass pollen (granulocyte/pollen-binding protein [GPBP]). GPBP was purified by gel filtration, anion exchange, and affinity chromatography. Virtually all of the granulocyte/pollen-binding activity was associated with a beta-1-protein having a molecular mass of approximately 77,000 D and an isoelectric point of between 5.5 and 6.1. By immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was identified as transferrin. Monospecific antisera raised against either GPBP or transferrin removed biological activity from GPBP preparations, and GPBP and transferrin gave lines of identity with these two antisera. The apparent heterogeneity in the molecular size and charge of GPBP observed during progressive purification was minimal when GPBP was saturated with ferric ions before the separation procedures. These experiments indicate that granulocyte/pollen binding is a hitherto unrecognized property of transferrin which appears to be unrelated to iron transport and raises the possibility that transferrin might have a physiological role in the removal of certain organic matter.