Selective Accumulation Of Eosinophils Research Articles

Possible Mechanisms of Eosinophil Accumulation in Eosinophilic Pneumonia.

Eosinophilic pneumonia (EP), including acute EP and chronic EP, is characterized by the massive pulmonary infiltration of eosinophils into the lung. However, the mechanisms underlying the selective accumulation of eosinophils in EP have not yet been fully elucidated. We reported that bronchoalveolar lavage fluid (BALF) from EP patients induced the transmigration of eosinophils across endothelial cells in vitro. The concentrations of eotaxin-2 (CCL24) and monocyte chemotactic protein (MCP)-4 (CCL13), which are CC chemokine receptor (CCR) 3 ligands, were elevated in the BALF of EP patients, and anti-CCR3 monoclonal antibody inhibited the eosinophil transmigration induced by the BALF of EP patients. The concentration of macrophage inflammatory protein 1β (CCL4), a CCR5 ligand that induces eosinophil migration, was increased in the BALF of EP patients. Furthermore, the concentration of interleukin (IL) 5 was increased in the BALF of EP patients, and it has been reported that anti-IL-5 antibody treatment resulted in remission and the reduction of glucocorticoid use in some cases of chronic EP. The concentrations of lipid mediators, such as leukotriene (LT) B4, damage-associated molecular pattern molecules (DAMPs), such as uric acid, or extracellular matrix proteins, such as periostin, were also increased in the BALF of EP patients. These findings suggest that chemokines, such as CCR3/CCR5 ligands, cytokines, such as IL-5, lipid mediators, such as LTB4, DAMPs, and extracellular matrix proteins may play roles in the accumulation or activation of eosinophils in EP.

Eosinophil transendothelial migration induced by the bronchoalveolar lavage fluid of acute eosinophilic pneumonia.

Acute eosinophilic pneumonia (AEP) is characterized by a massive pulmonary infiltration of eosinophils. Mechanisms regulating the selective accumulation of eosinophils in AEP have not been fully established. The objective of this study was to evaluate the mechanisms of eosinophil accumulation in alveolar spaces through examination of bronchoalveolar lavage fluid (BALF) from AEP patients (AEP-BALF). Eosinophils were isolated from the blood of healthy subjects and were placed on a human pulmonary microvascular endothelial cell monolayer cultured on Transwell filters (Coster, Cambridge, MA, USA). A saline control solution or BALF from patients with AEP, sarcoidosis or hypersensitivity pneumonitis was applied to the lower compartment, and the transendothelial migration of the eosinophils was evaluated. The concentrations of cytokines and chemokines in BALF were also measured. Transmigration of eosinophils across endothelial cells was only induced by the AEP-BALF. This transmigration was blocked by anti-β2 integrin mAb. The concentrations of eotaxin-2 and monocyte chemotactic protein (MCP)-4, which are CC chemokine receptor (CCR) 3 ligands, were elevated in the AEP-BALF, and anti-CCR3 mAb or anti-MCP-4 mAb inhibited the AEP-BALF-induced transmigration of eosinophils. Furthermore, the concentration of leukotriene (LT) B4 was increased in the AEP-BALF, and an LTB4 receptor antagonist partially suppressed the AEP-BALF-induced transmigration of eosinophils. These findings suggest that CCR3 ligands including eotaxin-2 and MCP-4, and LTB4 play a role in the accumulation of eosinophils in AEP.

Eotaxin-1 in exhaled breath condensate of stable and unstable asthma patients.

BackgroundAirway eosinophilia is considered a central event in the pathogenesis of asthma. Eotaxin plays a key role in selective eosinophil accumulation in the airways and, subsequently, their activation and degranulation. The study was undertaken to evaluate eotaxin-1 levels in the exhaled breath condensate (EBC) of asthmatics with different degrees of asthma severity and to establish the possible correlation of these measurements with other recognized parameters of airway inflammation.MethodsEBC was collected from 46 patients with allergic asthma (14 with steroid-naïve asthma, 16 with ICS-treated, stable asthma, 16 with ICS-treated unstable asthma) and 12 healthy volunteers. Concentrations of eotaxin-1 were measured by ELISA.ResultsIn the three groups of asthmatics, eotaxin-1 concentrations in EBC were significantly higher compared with healthy volunteers (steroid-naïve asthma: 9.70 pg/ml ± 1.70, stable ICS-treated asthma: 10.45 ± 2.00, unstable ICS-treated asthma: 17.97 ± 3.60, healthy volunteers: 6.24 ± 0.70). Eotaxin-1 levels were significantly higher in patients with unstable asthma than in the two groups with stable disease. We observed statistically significant correlations between the concentrations of eotaxin-1 in EBC and exhaled nitric oxide (FENO) or serum eosinophil cationic protein (ECP) in the three studied groups of asthmatics. We also discovered a significantly positive correlation between eotaxin-1 in EBC and blood eosinophil count in the groups of patients with unstable asthma and steroid-naïve asthma.ConclusionsMeasurements of eotaxin-1 in the EBC of asthma patients may provide another useful diagnostic tool for detecting and monitoring airway inflammation and disease severity.

Leukotriene B 4 is essential for selective eosinophil recruitment following allergen challenge of CD4 + cells in a model of chronic eosinophilic inflammation

Subcutaneous heat-coagulated egg white implants (EWI) induce chronic, intense local eosinophilia in mice, followed by asthma-like responses to airway ovalbumin challenge. Our goal was to define the mechanisms of selective eosinophil accumulation in the EWI model. EWI carriers were challenged i.p. with ovalbumin and the contributions of cellular immunity and inflammatory mediators to the resulting leukocyte accumulation were defined through cell transfer and pharmacological inhibition protocols. Eosinophil recruitment required Major Histocompatibility Complex Class II expression, and was abolished by the leukotriene B4 (LTB4) receptor antagonist CP 105.696, the 5-lipoxygenase inhibitor BWA4C and the 5-lipoxygenase activating protein inhibitor MK886. Eosinophil recruitment in EWI carriers followed transfer of: a) CD4 + (but not CD4 −) cells, harvested from EWI donors and restimulated ex vivo; b) their cell-free supernatants, containing LTB4. Restimulation in the presence of MK886 was ineffective. CC chemokine receptor ligand (CCL)5 and CCL2 were induced by ovalbumin challenge in vivo. mRNA for CCL17 and CCL11 was induced in ovalbumin-restimulated CD4 + cells ex vivo. MK886 blocked induction of CCL17. Pretreatment of EWI carriers with MK886 eliminated the effectiveness of exogenously administered CCL11, CCL2 and CCL5. In conclusion, chemokine-producing, ovalbumin-restimulated CD4 + cells initiate eosinophil recruitment which is strictly dependent on LTB4 production.

Eosinophil trafficking: new answers to old questions

Eosinophil trafficking: new answers to old questions

Human Platelets Produce Granulocyte-Macrophage Colony-Stimulating Factor and Delay Eosinophil Apoptosis

An association between eosinophils and platelets has been described in several diseases, most notably asthma. Although the mechanisms through which platelets influence eosinophil behavior are not well defined, platelets seem to contribute to the selective accumulation of eosinophils at sites of allergic inflammation by virtue of their ability to produce eosinophil chemotactic factors. We report here for the first time that platelets delay apoptosis, thus enhancing eosinophil survival. A marked inhibition of spontaneous apoptosis was observed using eosinophil:platelet ratios of 1:50, 1:25, 1:10, and 1:5. Moreover, promotion of eosinophil apoptosis by either pronase or dexamethasone was also inhibited greatly in the presence of platelets. The antiapoptotic effect mediated by platelets was dependent on the release of soluble products and was significantly inhibited by neutralizing antibodies directed to GM-CSF. Studies performed by flow cytometry, directed to analyze the cellular source of this cytokine, demonstrated that intracytoplasmic GM-CSF is present in resting platelets. Moreover, GM-CSF was found in platelet supernatants, at concentrations able to prevent eosinophil apoptosis. Our findings support a novel mechanism through which platelets may contribute to eosinophil accumulation at allergic inflammatory sites.

Eosinophil Transmigration across VCAM-1-Expressing Endothelial Cells Is Upregulated by Antigen-Stimulated Mononuclear Cells

Background: Adhesion to and transmigration across endothelial cells expressing vascular cell adhesion molecule 1 (VCAM-1) may be key steps in the development of selective eosinophil accumulation at the allergic inflammation sites. There is evidence that cytokines/chemokines produced by CD4+ T cells play a prominent role in these processes. Objective: The objective of this study was to evaluate whether eosinophil migration across human pulmonary microvascular endothelial cells (HPMEC) expressing VCAM-1 is modulated by the supernatants of antigen-stimulated mononuclear cells obtained from atopic asthmatics. Methods: Peripheral blood mononuclear cells (PBMC) were isolated from Dermatophagoides farinae(Df)-sensitive asthmatic subjects and cultured for 96 h at 37°C in the presence or absence of 1 µg/ml Df antigen. Eosinophils were isolated from blood of healthy subjects and placed on the HPMEC monolayers cultured on a transwell filter (3-µm pore size) stimulated with IL-4 plus TNF-α (both at 100 pM, 24 h) The supernatants of PBMC were then applied to the lower compartment and the transmigration of eosinophils was examined. Results: The supernatants of PBMC stimulated with Df significantly enhanced the eosinophil transmigration across VCAM-1-expressing HPMEC (% migration: 7.6 ± 0.6 by the supernatants of PBMC cultured without Df vs. 12.3 ± 1.2 by the PBMC cultured with Df, p < 0.01, n = 8). The enhanced migration, but not spontaneous migration, was blocked by the anti-α₄ integrin antibody. Moreover, the enhanced transmigration was blocked by anti-CCR3 antibody. Conclusion: The antigen-stimulated PBMC from atopic asthmatics produce an activity to induce eosinophil migration across VCAM-1-expressing endothelial cells. This activity appears to involve CCR3 as an essential molecule.

Eosinophil recruitment into sites of delayed-type hypersensitivity reactions in mice

The selective accumulation of eosinophils in tissue is a characteristic feature of allergic diseases where there is a predominance of lymphocytes expressing a Th2 phenotype. In an attempt to define factors determining specific eosinophil accumulation in vivo, we have used a radiolabeled technique to assess the occurrence and the mechanisms underlying (111)In-eosinophil recruitment into Th1- and Th2-predominant, delayed-type hypersensitivity (DTH) reactions. Eosinophils were purified from the blood of IL-5 transgenic mice, labeled with (111)In and injected into nontransgenic CBA/Ca mice. Th1- and Th2-predominant, DTH reactions were induced in mice by immunization with methylated bovine serum albumin (MBSA) in Freund's complete adjuvant or with Schistosoma mansoni eggs, respectively. In these animals, (111)In-eosinophils were recruited in skin sites in an antigen-, time-, and concentration-dependent manner. Depletion of CD4+ lymphocytes abrogated (111)In-eosinophil recruitment in both reactions. Pretreatment of animals with anti-IFN-gamma mAb abrogated (111)In-eosinophil recruitment in MBSA-immunized and -challenged animals, whereas anti-IL-4 inhibited (111)In-eosinophil recruitment in both models. Local pretreatment with an anti-eotaxin polyclonal antibody inhibited the MBSA and SEA reactions by 51% and 39%, respectively. These results demonstrate that, although eosinophilia is not a feature of Th1-predominant, DTH reactions, these reactions produce the necessary chemoattractants and express the necessary cell adhesion molecules for eosinophil migration. The control of the circulating levels of eosinophils appears to be a most important strategy in determining tissue eosinophilia.

Interleukin-13 induces PSGL-1/P–selectin–dependent adhesion of eosinophils, but not neutrophils, to human umbilical vein endothelial cells under flow

Selective eosinophil accumulation is a hallmark of diseases such as asthma. In a model of chronic eosinophilic inflammation, we have previously shown that the tethering step in eosinophil adhesion is mediated by PSGL-1 binding to P-selectin. The Th2-associated cytokine IL-13 is of potential importance in allergic disease. We have therefore investigated whether IL-13 can mediate eosinophil binding to human umbilical vein endothelial cells (HUVEC) through P-selectin. IL-13 caused dose- and time-dependent increases of P-selectin expression, as assessed by flow and laser scanning cytometry. A similar degree of expression was observed with IL-4. There was no effect on E-selectin or ICAM-1 expression. Tumor necrosis factor- induced the expression of VCAM-1, E-selectin, and ICAM-1 but had no effect on P-selectin expression. IL-13 increased the production of mRNA for surface and soluble variants of P-selectin. Under flow conditions, eosinophils, but not neutrophils, showed enhanced binding to IL-13 and to IL-4–stimulated HUVEC compared to medium-cultured cells. Eosinophil adhesion was completely inhibited by a blocking monoclonal antibody against PSGL-1 and P-selectin. Anti–VLA-4 and anti–VCAM-1 antibodies inhibited binding to a lesser extent. Thus, at physiologic levels of expression induced by Th2 cytokines, P-selectin/PSGL-1 supported eosinophil but not neutrophil adhesion. This mechanism is likely to be a key event leading to the selective accumulation of eosinophils in allergic inflammation.

Interleukin-13 induces PSGL-1/P–selectin–dependent adhesion of eosinophils, but not neutrophils, to human umbilical vein endothelial cells under flow

AbstractSelective eosinophil accumulation is a hallmark of diseases such as asthma. In a model of chronic eosinophilic inflammation, we have previously shown that the tethering step in eosinophil adhesion is mediated by PSGL-1 binding to P-selectin. The Th2-associated cytokine IL-13 is of potential importance in allergic disease. We have therefore investigated whether IL-13 can mediate eosinophil binding to human umbilical vein endothelial cells (HUVEC) through P-selectin. IL-13 caused dose- and time-dependent increases of P-selectin expression, as assessed by flow and laser scanning cytometry. A similar degree of expression was observed with IL-4. There was no effect on E-selectin or ICAM-1 expression. Tumor necrosis factor-α induced the expression of VCAM-1, E-selectin, and ICAM-1 but had no effect on P-selectin expression. IL-13 increased the production of mRNA for surface and soluble variants of P-selectin. Under flow conditions, eosinophils, but not neutrophils, showed enhanced binding to IL-13 and to IL-4–stimulated HUVEC compared to medium-cultured cells. Eosinophil adhesion was completely inhibited by a blocking monoclonal antibody against PSGL-1 and P-selectin. Anti–VLA-4 and anti–VCAM-1 antibodies inhibited binding to a lesser extent. Thus, at physiologic levels of expression induced by Th2 cytokines, P-selectin/PSGL-1 supported eosinophil but not neutrophil adhesion. This mechanism is likely to be a key event leading to the selective accumulation of eosinophils in allergic inflammation.

Measurement of eosinophil accumulation in vivo.

Eosinophils are leukocytes that contain characteristic cationic proteins in their granules that bind the acidic dye eosin. In contrast to neutrophils, eosinophils are minority cells in the blood and are predominantly tissue-dwelling cells found at sites in contact with the environment: the mucosal surfaces of the lung, gastrointestinal tract, and genitourinary tract. Selective accumulation of eosinophils, as opposed to neutrophils, is one of the major pathological features of the inflammatory response to infection with parasitic helminths, and in several diseases such as asthma, allergic rhinitis, and atopic dermatitis. A key step in leukocyte recruitment is the local production of chemoattractant molecules that orchestrate the adhesive interactions between leukocytes and the vascular endothelium.

CD49d expression and function on allergen-stimulated T cells from blood and airway.

The alpha4 chain (CD49d), which constitutes one of the chains of alpha4beta1 (very late activating antigen-4 [VLA-4]) and alpha4beta7 integrins, mediates migration of T cells to extravascular spaces. The interaction between VLA-4 and vascular cell adhesion molecule-1 (VCAM-1) has been shown to be the critical pathway for the selective accumulation of eosinophils and basophils at sites of allergic inflammation. T lymphocytes are also specifically recruited into allergic sites, including the allergic asthmatic airway. Increased numbers of activated CD4+ cells expressing the DR antigen subset of the human leukocyte antigens (HLA-DR) appear in the allergic lung 48 h after allergen inhalation. The mechanisms by which these cells localize into the lung are still unknown. We report that stimulation of allergen-specific T cells with allergen in vitro resulted in enhanced expression of alpha4 chain (CD49d) as measured by receptor density on allergen-specific T-cell lines and T-cell clones. Kinetic studies showed that CD49d density was enhanced over a 24- to 48-h period in a time-dependent fashion, and was coordinately upregulated with HLA-DR expression. We also demonstrated that increased expression of CD49d on T-cell lines 24 h and 48 h after stimulation correlated with increased adhesion to the CS-1 fragment of fibronectin. In contrast, lymphocyte function-associated antigen-1b (LFA-1b) (CD11b), LFA-3 (CD58), and intercellular adhesion molecule-1 (ICAM-1) (CD54) expression did not change with allergen stimulation. We also showed that CD49d receptor density on T cells obtained by bronchoalveolar lavage (BAL) of allergic patients before and 48 h after allergen challenge was significantly higher than that on T cells taken from BAL of normal subjects and from controls with other inflammatory lung diseases. Taken together, these findings indicate that allergen stimulation activates allergen-specific T cells and coordinately induces increased CD49d receptor expression and binding to counterligands. We postulate that allergen-driven upregulation of CD49d, which together with the beta1 chain constitutes VLA-4 integrin, may be responsible for the selective accumulation of T cells in the allergic asthmatic lung.

The role of the eosinophil-selective chemokine, eotaxin, in allergic and non-allergic airways inflammation.

Blood eosinophilia and tissue infiltration by eosinophils are frequently observed in allergic inflammation and parasitic infections. This selective accumulation of eosinophils suggested the existence of endogenous eosinophil-selective chemoattractants. We have discovered a novel eosinophil-selective chemoattractant which we called eotaxin in an animal model of allergic airways disease. Eotaxin is generated in both allergic and non-allergic bronchopulmonary inflammation. The early increase in eotaxin paralleled eosinophil infiltration in the lung tissue in both models. An antibody to IL-5 suppressed lung eosinophilia, correlating with an inhibition of eosinophil release from bone marrow, without affecting eotaxin generation. This suggests that endogenous IL-5 is important for eosinophil migration but does not appear to be a stimulus for eotaxin production. Constitutive levels of eotaxin observed in guinea-pig lung may be responsible for the basal lung eosinophilia observed in this species. Allergen-induced eotaxin was present mainly in the epithelium and alveolar macrophages, as detected by immunostaining. In contrast there was no upregulation of eotaxin by the epithelial cells following the injection of sephadex beads and the alveolar macrophage and mononuclear cells surrounding the granuloma were the predominant positive staining cells. Eotaxin and related chemokines acting through the CCR3 receptor may play a major role in eosinophil recruitment in allergic inflammation and parasitic diseases and thus offer and attractive target for therapeutic intervention.

A role for lymphocytes and cytokines on the eosinophil migration induced by LPS.

In the present work we review the existing evidence for a LPS-induced cytokine-mediated eosinophil accumulation in a model of acute inflammation. Intrathoracic administration of LPS into rodents (mice, rats or guinea pigs) induces a significant increase in the number of eosinophils recovered from the pleural fluid 24 hr later. This phenomenon is preceded by a neutrophil influx and accompanied by lymphocyte and monocyte accumulation. The eosinophil accumulation induced by LPs is not affected by inhibitors of cyclo or lipoxygenase nor by PAF antagonists but can be blocked by dexamethasone or the protein synthesis inhibitor cycloheximide. Transfer of cell-free pleural wash from LPS injected rats (LPS-PW) to naive recipient animals induces a selective eosinophil accumulation within 24 hr. The eosinophilotactic activity present on the LPS-PW has a molecular weight ranging between 10 and 50 kDa and its effect is abolished by trypsin digestion of the pleural wash indicating the proteic nature of this activity. The production of the eosinophilotactic activity depends on the interaction between macrophages and T-lymphocytes and its effect can not be blocked by anti-IL-5 monoclonal antibodies. Accumulated evidence suggest that the eosinophil accumulation induced by LPS is a consequence of a eosinophilotactic cytokine produced through macrophage and T-cell interactions in the site of a LPS-induced inflammatory reaction.

Cytokine and eosinophil responses in the lung, peripheral blood, and bone marrow compartments in a murine model of allergen-induced airways inflammation.

Selective accumulation of eosinophils and activated CD4+ cells is now considered a central event in the pathogenesis of asthma, and this process is thought to be mediated by a number of cytokines including tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the Type 2 cytokines interleukin-4 (IL-4) and IL-5. To carry out a detailed time-course analysis of cellular changes in the bronchoalveolar lavage fluid (BAL), peripheral blood (PB), and bone marrow (BM), and of changes in the aforementioned cytokines in BAL and serum, Balb/c mice were sensitized by intraperitoneal injection with ovalbumin (OVA) adsorbed to aluminum hydroxide on two occasions 5 days apart, and were subjected to an OVA aerosol challenge 12 days after the second sensitization. This resulted in an airways inflammatory response characterized by early transient neutrophilia, marked eosinophilia, and, to a lesser extent, lymphocytosis in the BAL. Inflammatory events were first observed 3 h and 24 h after antigen challenge in the lung tissue and BAL, respectively, and lasted for 21 days. In the BM, we detected a 1.5- and 5-fold increase in the total number of cells and eosinophils, respectively, 4 days after the second sensitization. This was followed by a decrease, although BM eosinophilia remained clearly present at the time of antigen challenge. A second eosinopoietic event was observed in the BM shortly after challenge and reached a peak at day 3. BM cellularity returned to normal at day 21 after challenge. Serum OVA-specific IgE was first detected 3 days following the second sensitization (150 ng/ml). IgE levels then decreased but remained at the 75 ng/ml range at the time of the aerosol challenge. During the sensitization period, TNF-alpha (approximately 25 pg/ml), IL-4 (approximately 40 pg/ml), and IL-5 (approximately 250 pg/ml) were detected in serum, but not in the BAL fluid (BALF) and returned to background levels at the time of the antigen challenge. After antigen challenge, TNF-alpha, IL-4, IL-5, and GM-CSF were detected in serum. Peak levels were observed at 3 h (approximately 40 pg/ml), 3 h (approximately 120 pg/ml), 12 h (approximately 350 pg/ml), and 3 h (approximately 10 pg/ml), respectively, and returned to background levels 24 h after challenge. In the BALF, we detected peak levels of TNF-alpha, IL-4, IL-5, and GM-CSF at 6 h (approximately 250 pg/ml), 24 h (approximately 140 pg/ml), 24 h (350 pg/ml), and 3 h (approximately 10 pg/ml), respectively, with a return to background levels 5 days after challenge. No IL-10 could be detected at any time point during sensitization or after challenge in either serum or BAL. We also detected approximately 40 pg/ml of interferon-gamma (IFN-gamma) in the serum of normal untreated mice. Serum IFN-gamma levels fluctuated during sensitization and after challenge, but never exceeded those observed in untreated mice. Thus, the cytokine profile observed in this experimental model of allergic inflammation is characterized by IL-4 and IL-5 dominance, with an apparently minor TNF-alpha and GM-CSF contribution and relatively low or undetectable levels of IFN-gamma and IL-10.

Relationship between interleukin-5 and eotaxin in regulating blood and tissue eosinophilia in mice.

The mechanism of cooperation between IL-5 and eotaxin for the selective accumulation of eosinophils at sites of allergic inflammation is unknown. In this investigation we have used IL-5 deficient mice to define the relationship between this cytokine and eotaxin in the regulation of blood eosinophilia and eosinophil homing and tissue accumulation. Both IL-5 and eotaxin could independently induce a rapid and pronounced blood eosinophilia in wild type mice when administered systemically. In contrast, only eotaxin induced a pronounced blood eosinophilia in IL-5 deficient mice. The eosinophilic response induced by intravenous eotaxin in wild type mice did not correlate with a significant reduction in the level of bone marrow eosinophils, whereas intravenous IL-5 resulted in depletion of this store. These results suggest the existence of two mechanisms by which eosinophils can be rapidly mobilized in response to intravenous eosinophil chemoattractants; first, mobilization of an IL-5 dependent bone marrow pool, and second, an eotaxin-induced sequestration of eosinophils from tissues into the blood. Subcutaneous injection of eotaxin induced a local tissue eosinophilia in wild type mice but not in IL-5 deficient mice. Furthermore, tissue eosinophilia in wild type mice, but not in IL-5 deficient mice, was enhanced by adoptive transfer of eosinophils or the administration of intravenous IL-5. However, pretreatment of IL-5 deficient mice with intraperitoneal IL-5 for 72 h restored eosinophil homing and tissue accumulation in response to subcutaneous eotaxin. We propose that eotaxin secreted from inflamed tissue may play an important role in initiating both blood and tissue eosinophilia in the early phases of allergic inflammation. Furthermore, IL-5 is not only essential for mobilizing eosinophils from the bone marrow during allergic inflammation, but also plays a critical role in regulating eosinophil homing and migration into tissues in response to eotaxin and possibly other specific chemotactic stimuli.

Cyclophosphamide-induced blood and tissue eosinophilia in contact sensitivity: mechanism of hapten-induced eosinophil recruitment into the skin.

The mechanism leading to selective production and accumulation of eosinophils in certain allergic skin diseases is unknown. Cyclophosphamide treatment (150 mg/kg) of BALB/c mice 48 h before sensitization with picryl chloride (PCl) resulted in striking blood and tissue eosinophilia, maximal at 13 days. Blood eosinophilia was not induced by the sensitization with oxazolone and 2,4-dinitrofluorobenzene. Challenge with 1 % PCl, but not croton oil caused preferential eosinophil accumulation into the dermis, which was associated with the enhanced expression of vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells. Intravenous administration of anti-VCAM-1 monoclonal antibody abrogated eosinophil infiltration. In this murine model, we examined the role of several cytokines, including chemokines in inducing selective tissue eosinophilia in vivo. Local administration of antibodies against interleukin (IL)-1beta, IL-4, tumor necrosis factor (TNF)-alpha, and RANTES, but not against IL-5 before challenge inhibited hapten-induced eosinophil recruitment. Intradermal injection of recombinant (r)IL-1beta, rIL-4, rTNF-alpha, rRANTES, and rMIP-1alpha induced marked eosinophil accumulation. Nonetheless, intradermal rIL-5 was not a chemoattractant for eosinophils in vivo. Our findings suggest that IL-1beta, IL-4, TNF-alpha, and RANTES contribute to the selective accumulation of eosinophils in contact sensitivity reaction. Although circulating IL-5 can activate eosinophils and prolong their survival, locally secreted IL-5 is not crucial for inducing eosinophil recruitment into the skin.

Increased adhesion to vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 of eosinophils from patients with asthma

Adhesion of peripheral blood eosinophil and neutrophil granulocytes to the endothelial cell adherence receptors E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 has been measured. The study included patients with allergic rhinitis, patients with mild allergic and nonallergic asthma, and healthy individuals; 10 persons were in each group. In addition, assay of eosinophil and neutrophil cell surface expression of the receptor complex CD11b/CD18 was performed. Increased eosinophil adhesion to vascular cell adhesion molecule-1 ( p < 0.05) and intercellular adhesion molecule-1 ( p < 0.05) was demonstrated in the patients with a more labile asthma, that is, a peak expiratory flow rate variability of more than 10%, suggesting a relationship to the degree of ongoing inflammation in the airways of the patients. The increased eosinophil adhesion was most probably due to a functional upregulation of the CD11b/CD18 and very late activation antigen-4 receptors, because the number of receptors measured as cell surface expression was unaltered. The increased eosinophil adhesion in the patients with high peak expiratory flow rate variability appeared independent of atopy. The increased adhesion was not entirely specific to the eosinophils, because neutrophils from patients with a peak expiratory flow rate variability of more than 10% also demonstrated increased adhesion to intercellular adhesion molecule-1 ( p < 0.05) when compared with neutrophils from the patients with low peak expiratory flow rate variability. In conclusion, the demonstrated priming of eosinophil adhesion to vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 might be one contributing mechanism behind the selective accumulation of eosinophils in the lung tissue of patients with asthma. (J A LLERGY C LIN I MMUNOL 1995;96:941-50.)

Eosinophil adhesion to vascular cell adhesion molecule-1 activates superoxide anion generation.

Adhesion to the adhesion protein, VCAM-1, on vascular endothelium is proposed to be an important factor in the selective accumulation of eosinophils at sites of allergic inflammation. To determine whether eosinophil adhesion to VCAM-1 is also associated with an alteration of eosinophil function, human peripheral blood eosinophils were isolated from allergic donors and incubated in VCAM-1-coated wells. Spontaneous adherence of isolated eosinophils to VCAM-1-coated wells was greater than cells incubated in FCS-treated control wells (38.0 +/- 1.6% vs 17.1 +/- 1.9%, n = 16, p < 0.0001). In addition, eosinophils incubated in VCAM-1-coated wells spontaneously generated modest but significant amounts of superoxide anion (O2-; 2.0 +/- 1.3 vs 00.5 +/- 0.5 nmol/5 x 10(5) cells, n = 9, p = 0.029). Moreover, when 100 nM FMLP was added to eosinophils in the presence of VCAM-1, significantly greater O2- generation occurred (7.2 +/- 0.9 vs 5.4 +/- 1.0 (FCS control) nmol/5 x 10(5) cells, n = 9, p = 0.009). Adhesion, as well as the spontaneous and enhanced O2- generation to FMLP activation, was blocked by the monoclonal anti-alpha 4 integrin Ab, HP 1/2, implying involvement of an alpha 4 integrin-VCAM-1 interaction. In contrast, the anti-CD18 mAb, L130, inhibited the spontaneous and enhanced O2- generation to FMLP without affecting adhesion, suggesting an involvement of CD18 molecule(s) only in VCAM-1-enhanced respiratory burst. Finally, 1 microM genistein, a tyrosine kinase inhibitor suppressed the VCAM-1-enhancing effect on eosinophil O2- generation and VCAM-1-induced tyrosine phosphorylation, suggesting a role for tyrosine phosphorylation in this eosinophil functional up-regulation. Our observations suggest that eosinophil adhesion to VCAM-1 may be an important step in determining the eventual functional activity of these cells as they migrate from the circulation to the airways and contribute to the allergic inflammatory process.

The Chemokine, Eotaxin, Activates Guinea-Pig Eosinophils in Vitro and Causes Their Accumulation into the Lung in Vivo

Eotaxin is a novel C-C chemokine purified from the bronchoalveolar lavage (BAL) fluid of actively sensitised guinea-pigs after aerosol allergen challenge. In this study we show that eotaxin induced a dose-dependent increase in both intracellular-free calcium concentration ([Ca2+]i) and aggregation of guinea-pig eosinophils in vitro. Intradermally injected eotaxin induced the accumulation of [111In]eosinophils in naive guinea-pig skin in vivo, without oedema-inducing activity; the latter emerging in separate BAL fluid HPLC fractions. Aerosol exposure of naive guinea-pigs to eotaxin in vivo caused a selective increase in eosinophils in BAL fluid. Thus, eotaxin activates guinea-pig eosinophils in vitro and causes a selective eosinophil accumulation in the lung in vivo. Eotaxin and related molecules are potentially important endogenous signalling substances in allergic reactions in vivo.