Neutrophils are important effector cells in immunity to microorganisms, particularly bacteria. Here, we show that the process of neutrophil apoptosis is delayed in several inflammatory diseases, suggesting that this phenomenon may represent a general feature contributing to the development of neutrophilia, and, therefore, in many cases to host defense against infection. The delay of neutrophil apoptosis was associated with markedly reduced levels of Bax, a pro-apoptotic member of the Bcl-2 family. Such Bax-deficient cells were also observed upon stimulation of normal neutrophils with cytokines present at sites of neutrophilic inflammation, such as granulocyte and granulocyte-macrophage colony-stimulating factors, in vitro. Moreover, Bax-deficient neutrophils generated by using Bax antisense oligodeoxynucleotides demonstrated delayed apoptosis, providing direct evidence for a role of Bax as a pro-apoptotic molecule in these cells. Interestingly, the Bax gene was reexpressed in Bax-deficient neutrophils under conditions of cytokine withdrawal. Thus, both granulocyte expansion and the resolution of inflammation appear to be regulated by the expression of the Bax gene in neutrophils.

A subgroup of patients with atopic dermatitis are known to have normal serum total immunoglobulin E levels, undetectable specific immunoglobulin E, and negative skin prick tests towards allergens. This form of the disease has been termed nonallergic atopic dermatitis. In this study, we found that, among 1151 chronic atopic dermatitis patients, about 10% had normal serum immunoglobulin E levels with no evidence for immunoglobulin E sensitization. We investigated immunologic mechanisms of patients with "allergic" and "nonallergic" atopic dermatitis using peripheral blood and skin biopsy samples. Our data suggest that T cells are likely involved in the pathogenesis of both forms of atopic dermatitis. Skin T cells equally responded to superantigen, staphylococcal enterotoxin B, and produced interleukin-2, interleukin-5, interleukin-13, and interferon-gamma in both forms of the disease. Interleukin-4, however, was not detectable in the skin biopsies of both atopic dermatitis types and was secreted in very low amounts by T cells cultured from the skin biopsies. Moreover, skin T cells from nonallergic atopic dermatitis patients expressed lower interleukin-5 and interleukin-13 levels compared with allergic atopic dermatitis patients. Accordingly, T cells isolated from skin biopsies of atopic dermatitis, but not from the nonallergic atopic dermatitis, induced high immunoglobulin E production in cocultures with normal B cells that was mediated by interleukin-13. In addition, B cell activation with high CD23 expression was observed in the peripheral blood of atopic dermatitis, but not nonallergic atopic dermatitis patients. These data suggest, although high numbers of T cells are present in lesional skin of both types, a lack of interleukin-13-induced B cell activation and consequent immunoglobulin E production in nonallergic atopic dermatitis.

To the Editor: In their report, Dibbert et al[1][1] had described that spontaneous eosinophil apoptosis was associated with a decrease in the levels of the antiapoptotic Bcl-XL protein. Because they also observed that granulocyte-macrophage colony-stimulating factor or interleukin-5 could maintain

It has been suggested that Fas ligand–Fas receptor interactions are involved in the regulation of eosinophil apoptosis and that dysfunctions in this system could contribute to the accumulation of these cells in allergic and asthmatic diseases. Here, we demonstrate that nitric oxide (NO) specifically prevents Fas receptor–mediated apoptosis in freshly isolated human eosinophils. In contrast, rapid acceleration of eosinophil apoptosis by activation of the Fas receptor occurs in the presence of eosinophil hematopoietins. Analysis of the intracellular mechanisms revealed that NO disrupts Fas receptor–mediated signaling events at the level of, or proximal to, Jun kinase (JNK), but distal to sphingomyelinase (SMase) activation and ceramide generation. In addition, activation of SMase occurs downstream of an interleukin 1 converting enzyme–like (ICE-like) protease(s) that is not blocked by NO. However, NO prevents activation of a protease that targets lamin B1. These findings suggest a role for an additional NO-sensitive apoptotic signaling pathway that amplifies the proteolytic cascade initialized by activation of the Fas receptor. Therefore, NO concentrations within allergic inflammatory sites may be important in determining whether an eosinophil survives or undergoes apoptosis upon Fas ligand stimulation.

The hypereosinophilic syndrome (HES) represents a heterogenous group of disorders characterized by hypereosinophilia in the blood and bone marrow, as well as eosinophilic tissue infiltration. The mechanisms of eosinophilic accumulation in HES are largely unknown. It has been recently suggested that the tissue eosinophilia observed in allergic disorders is, at least partially, caused by inhibition of eosinophil apoptosis by T cell–, mast cell–, and eosinophil-derived cytokines.1Simon HU Yousefi S Schranz C Schapowal A Bachert C Blaser K. Direct demonstration of delayed eosinophil apoptosis as a mechanism causing tissue eosinophilia.J Immunol. 1997; 158: 3902-3908PubMed Google Scholar Therefore we hypothesized that abnormalities in the process of eosinophil apoptosis may play a part in the etiology of HES. Members of the B-cell lymphoma-2 (Bcl-2) family of proteins are important regulators of apoptosis in many cellular systems.2Yang E Korsmeyer SJ. Molecular thanatopsis: a discourse on the BCL2 family and cell death.Blood. 1996; 88: 386-401Crossref PubMed Google Scholar The first member of the family, Bcl-2, was originally cloned from the breakpoint of a t(14;18) translocation present in many human B-cell lymphomas. Increased production of Bcl-2 protein as a result of t(14;18) translocations contributes to neoplastic B-cell expansion by preventing B-cell death.2Yang E Korsmeyer SJ. Molecular thanatopsis: a discourse on the BCL2 family and cell death.Blood. 1996; 88: 386-401Crossref PubMed Google Scholar In this article we describe an unusual case of HES with blood and tissue eosinophils that expressed, in contrast to normal eosinophils, the Bcl-2 gene. Because it was impossible to induce the Bcl-2 gene by IL-5 or GM-CSF in normal eosinophils, it is unlikely that the observed eosinophil Bcl-2 expression in this subject was the result of stimulation with eosinophil hematopoietins in vivo. Moreover, overexpression of Bcl-2 was associated with delayed death of blood eosinophils in vitro, suggesting that Bcl-2 did act as an apoptosis repressor in this cellular system. A 61-year-old woman had recurrent bilateral periorbital swelling and intermittent urticaria with annular erythematous skin lesions for more than 8 years. The laboratory findings included a marked persistent blood eosinophilia. The absolute eosinophil numbers in blood during the time of these investigations ranged between 2079/mm3 (without glucocorticosteroid therapy) and 600/mm3 (with glucocorticosteroid therapy). Recently, diarrhea, nausea, and vomiting occurred sporadically, and the patient noticed an increased abdominal circumference with an associated weight gain of 10 kg. Furthermore, she felt swelling of the arms and legs together with a feeling of tenseness. The patient had no personal or family history of atopy, and no hypersensitivity reactions were detected in multiple allergy tests. Further laboratory tests did not provide evidence for a parasitic disease. At the time of admission, she had subfebrile temperature and dyspnea, and her vitality was progressively decreasing. The clinical examination revealed a positive undulation phenomenon and dullness of the sides, suggesting the presence of ascites, which was later confirmed by sonography. The cells found in the sterile ascites fluid were almost only eosinophils, suggesting a massive eosinophilic gastrointestinal inflammation. Radiographs of the chest revealed bilateral basal pleural effusions. Electrocardiography, echocardiography, gynecologic examinations, and ocular examinations were without pathologic results. The bone marrow was markedly infiltrated by eosinophils with suppression of neutrophil granulopoiesis. Anti-Bcl-2 and control IgG1 mAb were obtained from Dako (Zurich, Switzerland); FITC-conjugated anti-Bcl-2 mAb from Ancell Corp (Bayport, Minn); FITC-conjugated control IgG1 mAb from Coulter (Hialeah, Fla); anti-Fas receptor mAb (clone CH-11, IgM) from Immunotech (Marseille, Fance); and anti-CD16 mAb microbeads from Miltenyi Biotec (Bergisch-Gladbach, Germany). Eosinophils were purified as previously described.3Simon HU Yousefi S Dommann-Scherrer CC Zimmermann DR Bauer S Barandun J et al.Expansion of cytokine-producing CD4- CD8- T cells associated with abnormal Fas expression and hypereosinophilia.J Exp Med. 1996; 183: 1071-1082Crossref PubMed Scopus (140) Google Scholar, 4Hebestreit H Yousefi S Balatti I Weber M Crameri R Simon D et al.Expression and function of the Fas receptor on human blood and tissue eosinophils.Eur J Immunol. 1996; 26: 1775-1780Crossref PubMed Scopus (80) Google Scholar Eosinophils were cultured at 1 × 106 /mL for the indicated times with complete culture medium (RPMI 1640 supplemented with 10% FCS). GM-CSF was a kind gift from Dr Thomas Hartung (University of Konstanz, Konstanz, Germany). IL-5 was obtained from Genzyme (Cambridge, Mass). The cytokine concentrations were 25 ng/mL. Anti-Fas receptor mAb was used at 1 μg/mL. mRNA expression of Bcl-2 and Fas receptor was studied by using RT-PCR. Primers for Bcl-2 (5 ́-ACA ACA TCG CCC TGT GGA TGA C-3 ́ and 5 ́-ATA GCT GAT TCG ACG TTT TGC C-3 ́) and Fas receptor (5 ́-GTG GGA TCC CAC TTC GGA GGA TTG CTC AAC AAC C-3 ́ and 5 ́-GTG CTG CAG TAT GTT GGC TCT TCA GCG CTA ATA-3 ́) amplifications were synthesized (HSC Biotechnology Service Centre, Toronto, Ontario) according to previously published sequences.3Simon HU Yousefi S Dommann-Scherrer CC Zimmermann DR Bauer S Barandun J et al.Expansion of cytokine-producing CD4- CD8- T cells associated with abnormal Fas expression and hypereosinophilia.J Exp Med. 1996; 183: 1071-1082Crossref PubMed Scopus (140) Google Scholar, 4Hebestreit H Yousefi S Balatti I Weber M Crameri R Simon D et al.Expression and function of the Fas receptor on human blood and tissue eosinophils.Eur J Immunol. 1996; 26: 1775-1780Crossref PubMed Scopus (80) Google Scholar, 5Akbar AN Borthwick NJ Wickremasinghe RG Panayiotidis P Pilling D Bofill M et al.Interleukin-2 receptor common γ-chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL ) but not pro-apoptotic (bax, bcl-xS ) gene expression.Eur J Immunol. 1996; 26: 294-299Crossref PubMed Scopus (330) Google Scholar Primers for β-actin control amplification were obtained from Clontech (Palo Alto, Calif). For Bcl-2 amplification, cDNA from HL-60 cells was used as positive template DNA. This amplification product was confirmed by sequencing. In negative control experiments all PCR reagents for amplification were added except cDNA. After amplification, PCR products were run on 1% agarose gel and stained by ethidium bromide. Purified eosinophils (0.1 × 106 ) were stained with FITC-conjugated anti-Bcl-2 or control mAb for 15 minutes at room temperature after 45 minutes of permeabilization of the cell membrane with PermeaFix (Ortho Diagnostic Systems, Raritan, NJ). Stained cells were washed, fixed in PBS-buffered 2% paraformaldehyde solution, and analyzed by flow cytometry in an EPICS XL (Coulter). Immunohistochemistry was performed with skin biopsy specimens from the presented case and other patients with eosinophilic infiltration of the skin. Immunostaining was performed by using the alkaline phosphatase antialkaline phosphatase (APAAP) method with a commercial kit (Dako) according to the manufacturer’s instructions. Sections were slightly counterstained with hematoxylin, mounted, and examined under a Zeiss Axioscope at an original magnification of ×400. IL-5 concentrations in plasma were measured by solid-phase ELISA. Cell death of eosinophils was assessed by uptake of 1 μmol/L ethidium bromide and flow cytometric analysis (EPICS XL) as previously described.3Simon HU Yousefi S Dommann-Scherrer CC Zimmermann DR Bauer S Barandun J et al.Expansion of cytokine-producing CD4- CD8- T cells associated with abnormal Fas expression and hypereosinophilia.J Exp Med. 1996; 183: 1071-1082Crossref PubMed Scopus (140) Google Scholar, 4Hebestreit H Yousefi S Balatti I Weber M Crameri R Simon D et al.Expression and function of the Fas receptor on human blood and tissue eosinophils.Eur J Immunol. 1996; 26: 1775-1780Crossref PubMed Scopus (80) Google Scholar As demonstrated in Fig 1 (left panel), the purified eosinophils of the presented patient with HES demonstrated, compared with normal control eosinophils, a marked delay of eosinophil death in vitro. In contrast to normal eosinophils, the eosinophil viability did not appear to depend on the presence of an eosinophil hematopoietin, such as IL-5. However, stimulation of the Fas receptor by anti-Fas receptor mAb4Hebestreit H Yousefi S Balatti I Weber M Crameri R Simon D et al.Expression and function of the Fas receptor on human blood and tissue eosinophils.Eur J Immunol. 1996; 26: 1775-1780Crossref PubMed Scopus (80) Google Scholar induced normal death in these cells. Because Bcl-2 has been shown to prevent apoptosis in many cellular systems, we investigated the expression of this protein by HES and normal eosinophils. As shown in Fig 2, A and B , the HES, but not normal blood eosinophils, expressed significant levels of Bcl-2 mRNA and protein. Moreover, freshly purified blood neutrophils from normal control individuals (Fig 2, B ), as well as the presented patient with HES (not shown), did not express detectable Bcl-2 protein levels. In contrast to Bcl-2, no functional or expression abnormalities regarding the Fas receptor were observed in the HES eosinophils (Fig. 1, Fig. 2 ). To explore the mechanism of Bcl-2 upregulation, we first measured blood IL-5 levels. We observed a lower IL-5 concentration of the presented patient with HES compared with a patient with atopic dermatitis (0.61 vs 2.90 ng/mL). Although the IL-5 concentration was much higher in the patient with atopic dermatitis, no eosinophil Bcl-2 expression and a normal eosinophil death kinetic were observed (not shown). This suggested that increased IL-5 expression is not necessarily followed by increased Bcl-2 levels in eosinophils in vivo. To investigate more directly the effects of eosinophil hematopoietins on Bcl-2 expression in eosinophils, we incubated normal eosinophils with GM-CSF or IL-5. Neither cytokine induced Bcl-2 mRNA (Fig 2, C ) or protein (Fig 2, D ) in these cells, suggesting that eosinophil Bcl-2 expression is not regulated by eosinophil hematopoietins. These data were further confirmed by using additional techniques, such as Western blotting or immunocytochemistry (not shown). In addition, our results are in agreement with previously published work demonstrating low or no Bcl-2 expression in eosinophils,6Druilhe A Arock M Le Goff L Pretolani M. Human eosinophils express Bcl-2 family proteins: modulation of Mcl-1 expression by IFN-γ.Am J Respir Cell Mol Biol. 1998; 18: 315-322Crossref PubMed Scopus (52) Google Scholar as well as neutrophils.7Iwai K Miyawaki T Takizawa T Konno A Ohta K Yachie A et al.Differential expression of bcl-2 and susceptibility to anti-Fas-mediated cell death in peripheral blood lymphocytes, monocytes and neutrophils.Blood. 1994; 84: 1201-1208Crossref PubMed Google Scholar In contrast, another group observed the induction of the Bcl-2 gene after IL-5 stimulation in eosinophils.8Ochiai K Kagami M Matsumura R Tomioka H. IL-5 but not interferon-gamma (IFN-γ) inhibits eosinophil apoptosis by up-regulation of bcl-2 expression.Clin Exp Immunol. 1997; 107: 198-204Crossref PubMed Scopus (79) Google Scholar It is possible that differences in the methodology may have caused this discrepancy, although the reason is not clear. We also investigated the in vivo Bcl-2 expression by HES eosinophils by using eosinophil-infiltrated skin biopsy specimens and immunohistochemistry. As demonstrated in Fig 2, E , the HES eosinophils of the presented case provided evidence for Bcl-2 protein expression (76% of the tissue eosinophils were Bcl-2 positive), whereas the eosinophils of 3 other eosinophil-infiltrated skin biopsy specimens did not appear to express significant amounts of Bcl-2 protein (not shown). We also stained nasal polyp and eosinophilic cancer tissues and found no evidence for Bcl-2 expression by eosinophils in these models of tissue eosinophilia (not shown). After a 4-week period of glucocorticosteroid therapy with an initial dosage of 250 mg prednisone per day, the relative blood eosinophil count dropped from 60% to 18%. Moreover, under this therapy, the cutaneous manifestations and gastrointestinal symptoms were in complete remission. Interestingly, the prednisone treatment was also associated with a normalization of the patient’s eosinophil Bcl-2 protein expression (Fig 2, B ) and, consequently, of the in vitro eosinophil death response (Fig 1, right panel). These data further support the notion that the inhibited eosinophil death of the presented patient with HES was, at least partially, the consequence of increased expression of Bcl-2 mRNA and protein in these cells. Moreover, these data confirm previously published work1Simon HU Yousefi S Schranz C Schapowal A Bachert C Blaser K. Direct demonstration of delayed eosinophil apoptosis as a mechanism causing tissue eosinophilia.J Immunol. 1997; 158: 3902-3908PubMed Google Scholar on the important role of delayed eosinophil apoptosis for the development of eosinophilia in vivo. Further studies are needed to determine the molecular mechanisms responsible for the induction of the Bcl-2 gene in eosinophils. Regardless of the mechanism, Bcl-2 appears to play a role in the regulation of eosinophil apoptosis in at least some cases of HES. We thank Martina Weber for excellent technical assistance.

Fas ligand/Fas receptor molecular interactions have been implicated as having an important function for the regulation of eosinophil apoptosis. The purpose of the present study was to investigate biochemical events triggered by the engagement of the Fas receptor in freshly isolated human and mouse eosinophils. Activation of the Fas receptor on eosinophils with the agonistic anti-Fas monoclonal antibody (MoAb) resulted in increased tyrosine phosphorylation of several intracellular proteins. The tyrosine kinase inhibitors lavendustin A and genistein inhibited Fas receptor-induced cell death in both human and mouse eosinophils in vitro and prevented, at least partially, Fas receptor-mediated resolution of eosinophilic inflammation in a mouse in vivo model of lung eosinophilia. In addition, in freshly purified human eosinophils, lavendustin A prevented anti-Fas MoAb-induced proteolytic cleavage of lamin B, suggesting that tyrosine kinases may amplify the proteolytic signaling cascade within interleukin-1beta converting enzyme (ICE) family proteases. Moreover, the tyrosine kinase Lyn was identified as being involved in Fas receptor-mediated cell death. Collectively, these results demonstrate that tyrosine phosphorylation is an important step in the generation of the Fas receptor-linked transmembrane death signal in eosinophils and that Lyn participates in this pathway.

Eosinophils are potent inflammatory cells involved in allergic reactions. Inhibition of apoptosis of purified eosinophils by certain cytokines has been previously shown to be an important mechanism causing tissue eosinophilia. To elucidate the role of Bcl-2 family members in the inhibition of eosinophil apoptosis, we examined the expression of the known anti-apoptotic genes Bcl-2, Bcl-xL, and A1, as well as Bax and Bcl-xS, which promote apoptosis in other systems. We show herein that freshly isolated human eosinophils express significant amounts of Bcl-xL and Bax, but only little or no Bcl-2, Bcl-xS, or A1. As assessed by reverse transcription-polymerase chain reaction, immunoblotting, flow cytometry, and immunocytochemistry, we show that spontaneous eosinophil apoptosis is associated with a decrease in Bcl-xL mRNA and protein levels. In contrast, stimulation of the cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-5 (IL-5) results in maintenance or upregulation of Bcl-xL mRNA and protein levels. Moreover, Bcl-2 protein is not induced by GM-CSF or IL-5 in purified eosinophils. Bcl-2 protein is also not expressed in tissue eosinophils as assessed by immunohistochemistry using two different eosinophilic tissue models. Furthermore, Bcl-xL antisense but not scrambled phosphorothioate oligodeoxynucleotides can partially block the cytokine-mediated rescue of apoptotic death in these cells. These data suggest that Bcl-xL acts as an anti-apoptotic molecule in eosinophils.© 1998 by The American Society of Hematology.

Cytokine‐mediated inhibition of eosinophil apoptosis is a mechanism causing tissue eosinophilia. Previously published work suggested that activation of the Lyn‐Ras‐Raf‐1‐MAP kinase pathway is obligatory for prevention of eosinophil apoptosis by eosinophil hematopoietins. We demonstrate herein that activation of freshly isolated human blood eosinophils by granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) is associated with increased tyrosine phosphorylation of Jak2. The tyrosine kinase blocker, tyrphostin B42, prevented activation of Jak2 but not Lyn, suggesting that Jak2 is the specific target for tyrphostin B42 in eosinophils. In addition, since Lyn remained unaffected by tyrphostin B42, it is unlikely that Jak2 is required for Lyn activation in this model. To test whether tyrosine phosphorylation of Jak2 is linked to GM‐CSF‐mediated prolonged eosinophil survival, we determined the effect of tyrphostin B42 on eosinophil viability and apoptosis. Prevention of Jak2 activation by tyrphostin B42 was associated with the inability of GM‐CSF to prevent eosinophil apoptosis. These data suggest that disruption of not only the Lyn‐Ras‐Raf‐1‐MAP kinase but also the Jak‐STAT pathway blocks the ability of eosinophil survival factors to prevent apoptosis in eosinophils.