The mediation of tissue eosinophilia in hypersensitivity reactions. V. Comparative study of tissue eosinophilia in the skin lesions of local and systemic passive cutaneous anaphylactic reactions.

Tissue eosinophilia in active cutaneous anaphylaxis reactions is biphasic: the early phase (6 hr) is induced by a low molecular (mol. wt. 300) factor (early ECF), and the delayed phase (24 hr) is mediated by synergy of two different factors (delayed ECF-a and -b). In this study, we report the mediation of tissue eosinophilia in passive cutaneous anaphylactic (PCA) reaction sites. Tissue eosinophilia in systemic PCA showed two phases with peaks at 6 hr and 24 hr, while that in local PCA was monophasic and peaked at 12 hr. A dialysable eosinophil chemotactic factor was isolated from the early stage (0-6 hr) of local PCA skin sites, and another chemotactic factor (mol. wt. 15,000), sharing a common antigenicity with guinea-pig serum C5, from 12-hr-old local PCA skin sites. On the other hand, a different chemotactic factor with a mol. wt. of about 70,000, sharing a common antigenicity with delayed ECF-b isolated from active cutaneous anaphylactic skin lesions, was isolated from 24-hr-old systemic PCA skin lesions. Although the dialysable factor was also isolated from systemic PCA skin sites, the factor from systemic PCA delayed skin sites may not contribute to delayed tissue eosinophilia, since the activity paralleled the intensity of basophil accumulation but not to that of eosinophils. It is thus suggested that tissue eosinophilia in systemic and local PCA reactions is mediated by different chemotactic factors.     

The regulation of tissue eosinophil chemotactic factor and inhibitor in allergic skin lesions of Freund's complete adjuvant-treated guinea-pigs.

The regulation of tissue eosinophilia induced by dinitrophenyl-ascaris extract (DNP-As) was investigated in guinea-pigs. Biphasic tissue eosinophilia peaking at 6 and 24 hr was observed in the skin lesions in Bordetella pertussis vaccine (Bp)-treated animals. In contrast, only the early phase of tissue eosinophilia was observed in Freund's complete adjuvant (FCA)-treated animals. Although less eosinophil chemotactic activity was detected in 24-hr-old skin extract of FCA-treated animals (FCA-extract), evident activity was recovered in the concanavalin A eluate (Con A-eluate) when FCA-extract was fractionated by Con A Sepharose. The chemotactic factor in Con A-eluate of FCA-extract was confirmed to be the T cell-derived eosinophil chemotactic factor, termed Delayed ECF-a, which has been isolated from allergic skin lesions by immunoadsorption. Another factor from the same skin lesions, Delayed ECF-b (which is a serum-derived one), was not detected in the FCA-extract. When eosinophils were mixed or pretreated with Con A-effluent of FCA-extract, the treated cells failed to be attracted by Delayed ECF-a, while the response to Delayed ECF-b was not affected, indicating that the inhibition was selective for Delayed ECF-a but not for Delayed ECF-b, and the eosinophil chemotactic inhibitory factor (ECIF) acts on eosinophils directly. Major ECIF activity was associated with a mol. wt. of 70,000 and minor with 12,500. Furthermore, the activity was absorbed by eosinophils but not by macrophages suggesting that eosinophils have receptor sites for ECIF. It was thus suggested that the appearance of ECIF, which is selective for the response of eosinophils to Delayed ECF-a, and decreased Delayed ECF-b production resulted in the inhibition of delayed tissue eosinophilia in FCA-treated guinea-pigs.     

A novel eosinophil chemotactic factor derived from a histiocytic lymphoma of the central nervous system.

An eosinophil chemotactic activity was identified in extracts of tumour tissue and cerebrospinal fluid from a patient with a histiocytic lymphoma of the brain and spinal cord that was infiltrated extensively with eosinophils and associated with peripheral blood eosinophilia. The histiocytic lymphoma-derived eosinophils chemotactic factor, termed ECF-HL, exhibited a mol. wt of 13,000-14,000 by filtration on Sephadex G-50, was highly acidic based on its elution from a high pressure anion exchange column at pH 2 x 3 - 2 x 1, and was susceptible to inactivation by proteolytic digestion. ECF-HL was absent from extracts of control human brain tissue, glioblastomas, other lymphomas and a variety of carcinomas that lacked an accumulation of eosinophils. Partially purified ECF-HL had no chemokinetic activity, but rendered eosinophils unresponsive to other chemotactic factors. Thus ECF-HL is structurally and functionally distinct from other recognized peptides that are preferentially chemotactic for eosinophils.     

An eosinophilotactic factor derived from rat mast cells.

Rat eosinophils have been showed to respond chemotactically to the supernatant from mast cells which have been disrupted by freezing and thawing and from mast cells which have been specifically degranulated by Compound 48/80 or by antigen challenge or sensitised cells. The fact that the chemotactic response is not dependent on allergic degranulation suggests that the chemotactic factor is present pre-formed in the mast cells rather than being generated in response to an antigen-antibody reaction.     

The pathology of the autologous serum skin test response in chronic urticaria resembles IgE-mediated late-phase reactions

The wheal-and-flare response to intradermal autologous serum in chronic urticaria offers a model for study of the pathogenesis of the disorder. Serial biopsies of autologous serum induced wheals were performed in 5 chronic urticaria patients to assess the evolution of the cellular inflammatory response and to look for evidence of mast cell degranulation. Perivascular neutrophils and eosinophils were seen as early as 30 min, becoming more intense and diffuse over 2 h. T lymphocyte numbers were increased by 2 h, CD4+ cells outnumbering CD8+ cells at 24 h. By 48 h, the neutrophils were clearing, but eosinophils and lymphocytes persisted. The histology of compound 48/80-induced wheals was similar to serum-induced wheals, but there was little or no response to physiological saline (0.16 M). Stainable mast cells were reduced in compound 48/80- and serum-induced wheals when compared to saline skin tests. Mast cell granules appeared swollen and had lost their characteristic lamellar substructure on electron microscopy of a serum-induced wheal biopsied at 10 min. Eosinophil degranulation was also observed at 2 h. The resemblance of the inflammation to the late phase of IgE-mediated immediate hypersensitivity reactions in atopics supports the concept that a circulating factor causes mast cell degranulation in chronic urticaria and may be important in the pathogenesis of the disorder.     

The mediation of tissue eosinophilia in hypersensitivity reactions. II. Separation of a delayed eosinophil chemotactic factor from macrophage chemotactic factors.

In anaphylactic cutaneous lesions induced by DNP-ascaris extract in the guinea-pig, the time-course of delayed tissue eosinophilia was found to parallel that of the macrophage reaction, reaching its peak in 24 h. Macrophages could be differentiated from lymphocytes by the numerous lysosomal granules which stained for acid phosphatase. Extracts from such skin lesions contained a delayed eosinophil chemotactic factor and two different macrophage chemotactic factors. Most of the delayed eosinophil chemotactic factor was separated from the two macrophage chemotactic factors by gel filtration on Sephadex G-100 and Sephadex G-200 in that order. The eosinophil chemotactic factor after re-chromatography on Sephadex G-I99 showed no or little chemotactic activity for macrophages.     

Morphologic and functional evidence for release of mast-cell products in bullous pemphigoid.

We studied nine patients with bullous pemphigoid, a generalized cutaneous eruption- for evidence of mast-cell involvement during development of lesions. As in other reports, six of nine patients demonstrated a serum antibody directed against the epidermal basement-membrane zone. Direct immunofluorescence studies of lesions revealed depostion of immunoglobulin and complement proteins at the basement-membrane zone in six of nine and nine of nine patients, respectively. Participation of mast cells was suggested by a sequence of pathologic alterations in which there was progressive mast-cell degranulation and late eosinophil infiltration. In addition, a factor chemotactic for human eosinophils with the size and charge characteristics of the eosinophil chemotactic factor of anaphylaxis was identified in blullous fluid. The data indicate that, in addition to activation of the complement system, involvement of mast cells is an early and continuing event in the development of the cutanenous lesions of bullous pemphigoid.     

Human eosinophils, acidic tetrapeptides (ECF-A) and histamine. Interactions in vitro and in vivo.

The ECF-A acidic tetrapeptides Val-Gly-Ser-Glu, Ala-Gly-Ser-Glu and the analogue Val-Gly-Asp-Glu were selectively chemotactic for human eosinophils over a narrow dose range although eosinophils from different individuals varied in their dose-response pattern. Histamine abrogated the chemotactic properties of the individual tetrapeptides. When Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu were combined in various concentrations the resultant chemotaxis was either negligible or no greater than that produced when each peptide was tested separately. Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu both promoted eosinophil accumulation when applied to the abraded skin of man or i.d. to the marmoset. Biopsies of marmoset skin revealed that peptide-induced eosinophilia was not associated with mast-cell degranulation. Histamine, which was chemotactic in vitro, did not lead to appreciable eosinophil accumulation in vivo, and combinations of histamine and the acidic tetrapeptides evoked little or no cutaneous eosinophil infiltration either in man or the marmoset. These studies suggest that there is a complex interaction between histamine and the ECF-A tetrapeptides; however, the tetrapeptides alone can promote the recruitment and localization of eosinophils by a mechanism apparently independent of mast-cell degranulation.     

The mediation of tissue eosinophilia in hypersensitivity reactions. III. Separation of two different delayed eosinophil chemotactic factors and transfer of these factors by serum or cells from sensitized guinea-pigs.

Two different eosinophil chemotactic factors (delayed ECF-a and ECF-b) were isolated from the delayed phase (24 hr old) of eosinophil-rich inflammatory reaction sites induced by DNP-ascaris extract, which could be separated by DEAE-Sephadex. Both the factors were thermolabile (56 degrees, 30 min) and had a mol. wt of about 70,000, whereas they were clearly different in antigenicity. Delayed tissue eosinophilia was shown to be mediated by peritoneal exudate cells (PEC) and serum from immunized donors to recipients. A chemotactic factor, closely resembling a delayed ECF-a, was isolated from the skin lesions of recipients transferred by sensitized PEC and challenged. Another factor, closely resembling a delayed ECF-b, was isolated from those of recipients transferred by immune serum and challenged. Furthermore, we have shown that delayed ECF-b, but not delayed ECF-a, shared a common antigenicity with some guinea-pig serum component. Thus we concluded that delayed tissue eosinophilia was mediated by the combined actions of two different factors, delayed ECF-a and ECF-b.     

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.     

Eosinophil chemotactic factors from granuloma of Kimura's disease, with special reference to T lymphocyte-derived factors

The granuloma of patients with Kimura's disease characterized by tissue and peripheral blood eosinophilia was reviewed with respect to eosinophil infiltration. An infiltrate of inflammatory cells with histiocytes and a sprinkling of eosinophils were observed in the fibrous stroma surrounding the newly formed vessels. Mast cells were rarely seen in the areas where eosinophils were grouped together. Three different eosinophil chemotactic factors (ECF) were isolated from the granulomas of Kimura's disease. They were termed as low molecular weight (LMW), intermediate molecular weight (IMW), and high molecular weight (HMW)-ECF according to the profile on gel filtration (LMW-ECF, about 500; IMW-ECF, about 12,500; HMW-ECF, 45,000-70,000). In terms of their activity when extracted from the granuloma, LMW-ECF and HMW-ECF seemed to be major natural mediators for the tissue eosinophilia, whereas IMW-ECF was a minor one. In an in vitro system, it was shown that granuloma lymphoid cells produce spontaneously at least two ECF having similar properties to LMW- and HMW-ECF, respectively. By analysis with monoclonal antibodies, granuloma T cells, probably OKT4-positive cells, were shown to be responsible for the production of those two ECF. It was thus suggested that prolonged synthesis of LMW- and HMW-ECF by OKT4-positive T cells plays a crucial role in the local eosinophilia of Kimura's disease.     

The natural mediator for PMN emigration in inflammation. VIII. Production of leucoegresin-like chemotactic factor in reversed passive Arthus reactions in rats.

The mediation of tissue neutrophilia in the reversed passive Arthus reactions in rats was studied on extract from the skin lesions. Approximately 55 per cent of the neutrophil chemotactic activity in the reactions exhibiting a maximal tissue neutrophilia seemed to be associated with a leucoeresin-like chemotactic factor which can be absorbed by an immunoadsorbent chromatography with anti-rat IgG antibody. On the other hand, the neutrophil chemotactic activity, comparable to most of the remaining chemotactic activity, was reduced in complement-depleted conditions in which the intensity of tissue neutrophilia in the reactions was moderately decreased, suggesting a possible involvement of complement-derived chemotactic factors.     

The mediation of tissue eosinophilia in hypersensitivity reactions. IV. Production of delayed eosinophil chemotactic factor-a by peritoneal exudate cells from sensitized guinea-pigs in vitro.

An eosinophil chemotactic factor (ECF) was produced in the cell-free culture supernatants (CFS) of peritoneal exudate cells (PEC) from guinea-pigs immunized with dinitrophenyl derivatives of ascaris extract (DNP-As), when stimulated by the antigen in vitro without activation by immune complexes. A 2 or 6 hr pulse of the antigen was sufficient for ECF production, whereas long time incubation (48 hr) was required for the production of a sufficient amount of the factor. Treatment of PEC by cycloheximide resulted in the reduction of ECF production, suggesting that protein synthesis is essential. Its generation appeared carrier-specific and the source of the factor is presumed to be lymphocytes, probably T lymphocyte. The factor with a molecular weight of 70,000 shared a common antigenicity with delayed eosinophil chemotactic factor-a (delayed ECF-a), which was isolated from the skin lesions showing delayed tissue eosinophilia in vivo.     

The experimental production of increased eosinophils in rat late-phase reactions.

The effects of peripheral eosinophilia on the intensity, kinetics and cellular characteristics of rat cutaneous late-phase reactions (LPR) have been investigated. Two distinct methods of inducing peripheral eosinophilia did not alter either the intensity or the kinetics of LPR produced by the intradermal injection of anti-IgE, Compound 48/80, or isolated mast cell granules. Hypereosinophilic animals exhibited increased numbers of tissue eosinophils in LPR tissue sites which correlated with the elevations in their respective peripheral eosinophil counts; however, the absolute number of eosinophils present, although significant, was not impressive (less than 10% of total). Our data suggest that, although rat LPR can be modulated to involve increased tissue eosinophils by increasing the numbers of peripheral blood eosinophils, no effects of these procedures on altering either the intensity or the kinetics of these reactions can be appreciated.     

Eosinophils in skin lesions of erythema multiforme

To investigate the controversy regarding the presence of eosinophils in skin lesions of erythema multiforme, we undertook a retrospective clinicopathologic study of 19 recent cases that fulfilled clinical and histopathologic criteria for the disease. At least a few eosinophils were observed in 13 of 19 cases, and in four cases there were more than three per high-power field, qualifying as "tissue eosinophilia." Immunofluorescence studies in three cases with eosinophils failed to show the linear basement membrane zone fluorescence characteristic of bullous pemphigoid. Giemsa stains revealed that mast cells were present in lesions both with and without eosinophils. The only clinical features that distinguished patients with tissue eosinophilia from those without were an older age of incidence and a longer duration of disease prior to biopsy. Drugs were implicated as a causative factor in some patients both with and without eosinophils, but all four patients with tissue eosinophilia were believed to have drug-induced disease. We conclude that eosinophils do occur in skin lesions of erythema multiforme and are occasionally numerous.     

Time course of cellular infiltration in the nasal mucosa during the immediate allergic reaction

In 23 patients with allergic rhinitis, biopsies of the nasal mucous membrane were taken at one of the following times after challenge of one nostril with allergen: 0 (baseline) (n = 7), 1/2 h (n = 6), 1 h (n = 5), and 2 h (n = 5). In the nostril stimulated by allergen there was a transient early phase influx of eosinophils while the numbers of stainable mast cells decreased, probably due to their degranulation. In the contralateral unstimulated nostril, there was no change in numbers of eosinophils but the numbers of stainable mast cells decreased. These results support the proposed role in allergic rhinitis of the mast cell and eosinophil, and suggest that the eosinophil may be a rapidly mobilized effector cell.     

Comparison of histological parameters for the diagnosis of eosinophilic oesophagitis versus gastro‐oesophageal reflux disease on oesophageal biopsy material

Aims: Eosinophil infiltration of the oesophageal epithelium is the cardinal pathomorphological finding in eosinophilic oesophagitis (EO), but gastro‐oesophageal reflux disease (GORD) is also associated with increased eosinophils. The aim was to compare histological parameters for the diagnosis of EO versus GORD on routinely taken biopsy specimens. Methods and results: One hundred and five routine biopsy specimens with EO (n = 62), GORD (n = 24) and probable EO (n = 19) from 74 patients (52 men, 22 women; mean age 43.7 years) were analysed for numbers of eosinophils, mast cells, degranulation and qualitative changes of oesophageal epithelium using immunohistochemistry with monoclonal antibodies against eosinophil peroxidase and eosinophil major basic protein and mast cell tryptase. Eosinophil infiltration was significantly higher in EO than in GORD both on haematoxylin and eosin staining (54.8 versus 9.1; P < 0.05) and immunohistochemistry (77.5 versus 24.7; P < 0.05). Eosinophil degranulation was significantly more intense in EO than in GORD (1.16 versus 0.41; P < 0.05). Furthermore, eosinophilia‐codependent secondary qualitative changes of squamous epithelium in EO were generally more extensive than those in GORD. Conclusions: Histological differential diagnosis of EO and GORD should be based on eosinophil counts, secondary morphological changes of eosinophils and oesophageal squamous epithelium, especially in cases suspicious of EO.     

The chemical mediation of delayed hypersensitivity skin reaction. II. Characterization of a macrophage-chemotactic factor from bovine gamma-globulin-induced skin reaction in guinea pigs.

Macrophage-chemotactic factors were extracted from delayed hypersensitivity skin lesions induced by bovine gamma-globulin in guinea pigs. The most active factor, MCFS--1, was highly purified and found to be a heat-labile protein with a molecular weight of 150,000 and to possess in vivo as well as in vitro activity. This factor was homogeneous during polyacrylamide gel electrophoresis, and the chemotactic activity was associated exclusively with this band. Further characterization revealed that its isoelectric point was 6.7 to 6.9 and made a single arc in the beta-globulin region with rabbit antiserums against guinea pig serum on immunoelectrophoresis. This factor seemed to be antigenically different from immunoglobulin G (IgG) by immunodiffusion and immunoadsorption. On the other hand, the chemotactic activity of MCFS-2 was adsorbed by neither anti-IgG nor anti MCFS-1 and that of euglobulin fraction was partially adsorbed by anti-IgG. These indicate the presence of at least three types of antigenically different chemotactic factors for macrophages in the extracts of delayed hypersensitivity skin lesions.     

Mechanisms of eosinophilia in mice infested with larval Haemaphysalis longicornis ticks.

Infestation of larval Haemaphysalis longicornis ticks induced a threefold increase of eosinophils in the peripheral blood of normal WBB6F1- +/+ mice 2 days after tick infestation. In genetically mast cell-deficient WBB6F1- W/Wv mice, a threefold increase of blood eosinophils was observed 6 days after the tick infestation. However, marked infiltration of eosinophils was detected in the tick infestation sites of the WBB6F1- +/+ mice but not the WBB6F1- W/Wv mice. When the mast cell deficiency of WBB6F1- W/Wv mice had been rescued locally by intradermal injections of WBB6F1- +/+ mouse-derived cultured mast cells, a rapid increase of blood eosinophils and tissue infiltration of eosinophils were revealed following tick infestation. The intravenous (i.v.) injection of immune spleen or lymph node cells obtained from WBB6F1- +/+ mice 10 days after tick infestation led to significant eosinophilia in naive recipient mice. Treatment with anti-Thy-1.2 or anti-CD4 monoclonal antibody (mAb) and complement (C) completely abolished the eosinophilia; the early response (2 days after tick challenge) is dependent on mast cells at the feeding site, and the late response (6 days after tick challenge) is dependent on T lymphocytes. Since amplified interleukin-5 (IL-5) cDNA was detectable in the spleen cells 4 days after tick infestation, the late response might be mediated by IL-5. The infiltration of eosinophils at the feeding site of skin appeared to be dependent on mast cells.     

Isolation of an eosinophil chemotactic lymphokine as a natural mediator for eosinophil chemotaxis from concanavalin A-induced skin reaction sites in guinea-pigs.

An intradermal injection with 20 micrograms concanavalin A (Con A) induce a marked tissue eosinophilia peaking at 24 h after the injection in guinea-pigs. Two different eosinophil chemotactic factors (ECFs) were isolated from the Con A-induced skin reaction sites. A non-dialysable ECF with mol. wt of about 70,000 closely resembled delayed ECF-a, which had been isolated from active cutaneous anaphylactic skin lesions and confirmed to be a product of T lymphocytes by antigenic stimulation, by virtue of the antigenicity, the chromatographic profiles on Sephadex G-100 and on DEAE-Sephadex, affinity to Con A-Sepharose, and other physicochemical properties. The activity of the factor paralleled the intensity of tissue eosinophilia, suggesting that the factor may function for the tissue eosinophilia as a natural mediator. Although another ECF with a low mol. wt (dialysable) was isolated from the same skin lesions, the dialysable factor may not contribute to the tissue eosinophilia because the activity of the factor paralleled the intensity of tissue basophilia but not that of eosinophilia.