Shiro Horie

Eosinophil active cytokines and surface analysis of eosinophils in Churg-Strauss syndrome.

There are few reports regarding the measurement of cytokines and surface analysis of eosinophils in Churg-Strauss syndrome (CSS). To examine the pathophysiology of CSS, concentrations of cytokines in serum and bronchoalveolar lavage fluid (BALF), and surface antigens on peripheral blood eosinophils were analyzed in five patients with CSS. Concentrations of cytokines (interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-3 (IL-3), interleukin-5 (IL-5) and granulocyte/macrophage colony stimulating factor (GM-CSF) were measured using ELISA. Surface antigens on eosinophils in peripheral blood were analyzed using flow cytometry. A concentration of interleukin-5 (IL-5) and TNF-alpha in serum was detected in five cases; however IL-1 beta, GM-CSF, and IL-3 were detected in 3 of 5, 2 of 5, and 1 of 5 patients, respectively. In BALF, TNF-alpha and IL-5 were detected in 2 of 3 and 1 of 3 patients, respectively; however, neither IL-1 beta, GM-CSF, nor IL-3 was detected in any. Newly expressed surface antigens such as CD25, CD4, and CD69 were observed on peripheral blood eosinophils in five cases. CD54 and HLA-DR were expressed in 4 of 5 and 3 of 5 patients, respectively. Eosinophils in peripheral blood are activated to various degrees, possibly depending on cytokine stimulation. This eosinophil activation may be related to the clinical stage of CSS.

Effects of intracellular cyclic AMP modulators on human eosinophil survival, degranulation and CD11b expression.

Background: Brochial asthma is characterized by infiltration of inflammatory cells such as lymphocytes and eosinophils. Theophylline is one of the most widely used drugs in the therapy of bronchial asthma, and phosphodiesterase (PDE) inhibition is thought to be an important mechanism of its anti–inflammatory actions. However, the detailed effects of PDE inhibition on eosinophils still remain unclear. Methods: Eosinophils in peripheral blood obtained from normal subjects and patients with mild off–season allergic rhinitis were purified using CD16 negative selection. The following effects of theophylline (nonselective PDE inhibitor), KF19514 (selective PDE IV inhibitor), mirlinone (selective PDE III inhibitor), procaterol (β2–adrenoceptor agonist) and N6, 2′–O–dibutyryladenosine 3′5′–cyclic monophosphate (dB–cAMP; AMP analogue) on eosinophils were examined: (1) survival in the presence of interleukin–5, (2) degranulation by granulocyte/macrophage colony–stimulating factor (GM–CSF) or platelet–activating factor (PAF), (3) CD11b expression under GM–CSF or PAF stimulation and (4) intracellular cAMP level. Results: Eosinophil survival was inhibited by theophilline, KF19514 or procaterol. GM–CSF– or PAF–induced degranulation was inhibited by theophylline, KF19514, procaterol or dB–cAMP. CD11b up–regulation by PAF was inhibited by theophylline, KF19514 or dB–cAMP, while GM–CSF–stimulated CD11b up–regulation was not significantly inhibited by any of the drugs tested. The levels of intracellular cAMP were increased by theophylline, KF19514 and procaterol. Conclusions: Intracellular cAMP is an important factor in the regulation of eosinophil biological functions. PDE IV inhibitors and β2–agonists are suggested to be useful for the treatment of bronchial asthma through inhibition of eosinophil effector function.

A Case of Pathophysiologic Study in Kimura's Disease: Measurement of Cytokines and Surface Analysis of Eosinophils

BACKGROUND Kimuras disease is a rare but distinctive eosinophilic inflammatory disorder of unknown etiology; few reported case studies have focused on the immunopathologic background of this unique disease. OBJECTIVE To define better the immunopathogenetic features of Kimuras disease, we attempted to quantitatively analyze values of cytokines and soluble interleukin-2 receptor (sIL-2R) in peripheral blood (PB), as well as perform surface immunophenotypic analysis of eosinophils from a Japanese patient with chronic relapsing Kimuras disease. RESULTS Granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha) and sIL-2R were elevated, and newly expressed antigens on eosinophils CD4, CD25, and HLA-DR were found to be involved in the pathophysiology of this disorder. CONCLUSIONS Kimuras disease may be a disease in which activated lymphocytes release cytokines, and these released cytokines, such as GM-CSF and TNF-alpha cause eosinophil activation. These processes may be related to the pathogenesis of this disorder.

Intercellular adhesion molecule-1 on eosinophils is involved in eosinophil protein X release induced by cytokines.

Recent evidence suggests that adhesion molecules play important roles in eosinophil functions such as degranulation and superoxide anion production. CD11b/CD18 (Mac‐1) and CD49d/CD29 (VLA‐4) are involved in eosinophil–endothelial adhesion through their counterligands, intercellular adhesion molecule‐1 (ICAM‐1; CD54) and vascular cell adhesion molecule‐1 (VCAM‐1), respectively. CD54 is also induced on eosinophils by cytokine stimulation. We hypothesized that CD54 on human eosinophils may participate in eosinophil degranulation. CD54 was induced on eosinophils by a combination of human recombinant granulocyte–macrophage colony‐stimulating factor (rGM‐CSF) and human recombinant tumour necrosis factor‐α (rTNF‐α) within 2 hr of incubation, as determined by flow cytometric analysis. Recombinant GM‐CSF alone induced a slight but significant CD54 expression on eosinophils. Release of eosinophil protein X, an indicator of eosinophil degranulation, was induced by rGM‐CSF and this effect was synergistically enhanced by adding rTNF‐α. To determine the role of newly expressed CD54 in eosinophil degranulation, a blocking assay was performed using monoclonal antibody (mAb) against CD54 and CD18. Anti‐CD18 mAb and anti‐CD54 mAb markedly inhibited eosinophil degranulation induced by rGM‐CSF or a combination of rGM‐CSF and rTNF‐α. On the other hand, anti‐CD54 mAb had little effect on rGM‐CSF‐ or rGM‐CSF/rTNF‐α‐induced adhesion of eosinophils, whereas anti‐CD18 mAb significantly inhibited eosinophil adhesion. These results indicate that CD54 on eosinophils plays an important role in the eosinophil degranulation and that eosinophils are capable of interacting with other β2 integrin‐positive cells.

Predominant implication of IL-5 in acute eosinophilic pneumonia : Comparison with chronic eosinophilic pneumonia

Background: Acute eosinophilic pneumonia (AEP) is a rare disease with unknown etiology. To examine pathophysiology of AEP we measured the cell number of eosinophils and eosinophil active cytokines in the peripheral blood and bronchoalveolar lavage fluid (BALF) of AEP patients and compared the levels with those measured in chronic eosinophilic pneumonia (CEP) patients. Methods: Cell number of eosinophils in peripheral blood and BALF from patients with AEP (n = 3) and CEP (n = 3) were measured. Eosinophil active cytokines in serum and BALF from the patients were measured using ELISA. Results: Eosinophil cell number in peripheral blood was 274–1,377/mm3 in AEP and 526–2,500/mm3 in CEP. The percentages of BALF eosinophils were high in AEP and CEP. Eosinophilia disappeared after methylprednisolone pulse therapy (1 g for 3 days) in AEP, however the cell number of eosinophils gradually increased after methylprednisolone pulse therapy and then spontaneously decreased to within normal range without any further medication. The concentrations of IL-5 in AEP were very high in serum and in BALF, however the concentrations in CEP were low in serum and BALF. Conclusion: AEP is a disease in which eosinophil active cytokine IL-5 is predominantly involved; CEP is not. The factors involving eosinophil infiltration to inflammatory loci differ between AEP and CEP.

Analysis of recombinant human tumour necrosis factor-α-induced CD4 expression on human eosinophils

We examined the hypothesis that one of the pro‐inflammatory cytokines, tumour necrosis factor‐α (TNF‐α), could induce expression of the adhesion molecule CD4 on human eosinophils. We further examined the effector function of CD4 and the mechanisms regulating CD4 expression.

Interferon-γ Increases CD62L Expression on Human Eosinophils

Background: L-selectin (CD62L) and Mac-1 (CD11b/CD18) play a crucial role in the infiltration of eosinophils. However, changes in CD62L and CD11b expression on eosinophils after stimulation with cytokines were little studied. Methods: Eosinophils in peripheral blood of healthy volunteers were purified and cultured with interleukin-5 (IL-5), granulocyte/macrophage colony-stimulating factor (GM-CSF) or interferon-γ (IFN-γ). After up to 24 h incubation, CD62L and CD11b expression were examined using flow cytometry. The effects of dexamethasone (Dex), cycloheximide (CHX) or theophylline on CD62L expression on IFN-γ-stimulated eosinophils were also studied. Results: IL-5 or GM-CSF downregulated CD62L and upregulated CD11b expression on eosinophils after 30 min stimulation. Conversely, IFN-γ upregulated CD62L after 12 h stimulation in a time- and dose-dependent manner, and had no effect on CD11b expression. Dex, CHX or theophylline dose-dependently decreased CD62L expression on IFN-γ-stimulated eosinophils. Conclusions: IFN-γ is a particular cytokine which can increase CD62L expression on circulating or infiltrated human eosinophils. It is suggested that protein synthesis and intracellular cAMP participate in the increase in L-selectin expression on IFN-γ-stimulated eosinophils.

Antisense Cdk2 kinase oligodeoxynucleotide inhibits ICAM-1 expression in murine cardiac allograft arteriopathy.

CARDIAC transplantation as a treatment for end-stage cardiac disease has been established. However, the problem of accelerated graft coronary disease in long-term survivors remains. Adhesion molecules play critical roles in graft arteriopathy; that is, intercellular adhesion molecule 1 (ICAM-1) is known to be one of the most important molecules in atherogenesis. Inhibition of arterial neointimal formation by antisense phosphorothioate oligodeoxynucleotide (ODN) directed to cell-cycle-regulatory genes after balloon angioplasty in the rat carotid injury model has been reported. The enzyme, cycline-dependent kinase (cdk) 2 kinase, plays a critical role in transition through the G1/S phase. The hemagglutination virus of Japan (HVJ) (liposome method) has been shown to increase the efficiency of cellular uptake of ODN. To test the hypothesis that inhibition of cdk2 kinase expression can inhibit ICAM-1 expression in graft arteries, we studied the effect of antisense cdk2 kinase ODN by intraluminal delivery using HVJ-liposome–mediated transfer. In this study, we demonstrate clearly that antisense cdk2 ODN treatment results in near-complete inhibition of ICAM-1 expression in neointimal formation in murine cardiac allografts.

Theophylline Inhibits TNF-α-Induced CD4 Expression on Human Eosinophils and CD4+ Eosinophil Migration

Background: Increasing evidence regarding asthma suggests that CD4+ cells are preferentially recruited to sites of bronchial inflammation. Interleukin (IL)-16 has been reported as playing an important role in the accumulation of CD4+ cells. We have shown that the CD4 molecule is expressed on normal human eosinophils by tumor necrosis factor (TNF)-α stimulation. Methods: We evaluated the effects of theophylline, KF19514 [a selective phosphodiesterase (PDE) IV inhibitor] and dexamethasone on CD4 expression on eosinophils and eosinophil migration in response to IL-16, a natural soluble ligand of the CD4 molecule. Results: The maximum eosinophil migration was observed when eosinophils were cultured with TNF-α at 10 ng/ml for 18 h and the concentration of IL-16 was 10 pg/ml. CD4+ eosinophil migration in response to IL-16 was mostly, if not fully, chemokinetic and this migration was significantly inhibited by Fab of anti-CD4 monoclonal antibody. Theophylline (10–4–10–3M), KF19514 (10–7–10–6M) and dexamethasone (10–8– 10–6M) significantly inhibited CD4 expression on eosinophils induced by TNF-α. Theophylline (10–3M) and KF19514 (10–6M) inhibited CD4+ eosinophil migratory responses induced by IL-16, but 10–6M dexamethasone did not. Theophylline and KF19514 augmented the intracellular adenosine-3′,5′-cyclic monophosphate (cAMP) concentration in eosinophils, suggesting modulation by cAMP of CD4 expression and eosinophil migration. Conclusions: These data suggest that TNF-α-induced CD4+ eosinophils may contribute to eosinophil migratory responses induced by IL-16. Theophylline and selective PDE IV inhibitor may prevent airway inflammation by downregulating CD4 expression on eosinophils and inhibiting eosinophil migration through CD4 and IL-16 interaction.

Sensitive diagnosis of cardiac allograft rejection by detection of cytokine transcription in situ

OBJECTIVE In situ transcription of cytokines which are important in the development of cardiac rejection has not been evaluated for diagnosing rejection. The objective was to evaluate the usefulness of in situ reverse transcriptase polymerase chain reaction (RT-PCR) for sensitive detection of acute cardiac rejection. METHODS We studied interferon (IFN)-gamma and interleukin (IL)-2 expression using immunohistochemistry and in situ RT-PCR in murine cardiac transplant models. Hearts were heterotopically transplanted (BALB/c to C3H/He) and some mice were not treated (n = 23); others were treated with anti-intercellular adhesion molecule (ICAM)-1 and anti-lymphocyte function associated antigen (LFA)-1 monoclonal antibodies (mAbs) (n = 23). Allografts were removed at days 1 to 7. For control, isografts were harvested at day 7 (n = 2). RESULTS Mice without treatment rejected allografts within 7 days, while all mAb-treated recipients accepted allografts for the same period. At day 1, allografts of both groups showed scattered myocardial cell infiltration which increased in non-treated allografts, but remained stable in mAb-treated grafts thereafter. In situ RT-PCR showed that IL-2 and IFN-gamma mRNA positive cells were present in non-treated allografts, while few mRNA positive cells were expressed in infiltrating cells in the mAb-treated allografts (IL-2, day 3: 88.8 +/- 28.3 vs. 7.2 +/- 6.4, p < 0.05, positive cells within 10 fields per section). However, immunohistochemistry could not reveal the difference at day 3. CONCLUSION In situ RT-PCR is a sensitive method for diagnosing acute rejection, and it reveals the characteristics of myocardial infiltrate cells to determine their role in the process of rejection.