Regulation of CD8 expression in mast cells by exogenous or endogenous nitric oxide.

We recently reported a novel CD8 molecule on rat alveolar macrophages and peritoneal mast cells (PMC). However, little is known about the regulation of CD8 expression and function on these cells. We investigated the regulation of CD8 expression on PMC by NO, because NO can regulate inflammatory responses and also because anti-CD8 Ab stimulates inducible NO synthase and NO production by PMC and alveolar macrophages. Ligation of CD8alpha on PMC with Ab (OX8) induced CD8alpha mRNA expression after 3-6 h, and flow cytometry demonstrated that OX8 treatment increased CD8alpha protein expression compared with PMC treated with isotype control IgG1. To test whether NO mediates the up-regulation of CD8alpha, we used the NO donor S-nitrosoglutathione (500 microM) and NO synthase inhibitors (N(G)-monomethyl-L-arginine and N(G)-nitro-L-arginine methyl ester; 100 microM). S-nitrosoglutathione up-regulated both mRNA and protein expression of CD8alpha in PMC compared with that in sham-treated cells, while NO synthase inhibitors down-regulated OX8 Ab-induced CD8alpha expression. To investigate how NO regulates CD8 expression on PMC, we examined the cGMP-dependent pathway using 8-bromo-cGMP (2 mM) and the guanylate cyclase inhibitor, 1H-oxadiazoloquinoxalin-1-one (20 microM). 8-Bromo-cGMP up-regulated CD8 expression, whereas 1H-oxadiazoloquinoxalin-1-one down-regulated its expression. Thus, ligation of CD8 up-regulates CD8 expression on PMC, a response mediated at least in part by NO through a cGMP-dependent pathway. The significance of this up-regulation of CD8alpha on mast cells (MC) is unclear, but since ligation of CD8 on MC with OX8 Ab can alter gene expression and mediator secretion, up-regulation of CD8 may enhance the MC response to natural ligation of this novel form of CD8.

[A case report of allergic fungal sinusitis caused by Penicillium sp. and Cladosporium sp].

We report a case of allergic fungal sinusitis (AFS) caused by Penicillium sp. and Cladosporium sp. in a 57-year-old man satisfying the following diagnostic criteria: (1) chronic rhinosinusitis revealed by computed tomographic scan, (2) Japanese cedar pollinosis for 3 years, (3) positivity for Penicillium sp. and Cladosporium sp. by a skin test, (4) increased immunoglobulin E (IgE) specific to these fungi, (5) increased total IgE, (6) nasal polyps with severe eosinophilic invasion, (7) allergic mucin revealed by histopathological examination, (8) fungal hyphae revealed by histopathological examination and (9) detection of Penicillium sp. and Cladosporium sp. revealed by fungi culture. The patient was treated by endoscopic sinus surgery. Four weeks after surgery, nasal polyps recurred, but his condition was improved by oral administration of steroids and nebulizer treatment with steroids and fluconazole. Total IgE, specific IgE and eosinophil count in the peripheral blood decreased, apparently reflecting this improvement. After obtaining the patient's consent, we conducted an allergen provocation test, which is as highly diagnostic as a skin test, to test for an antigen causing type I hypersensitivity. The immediate phase response was positive, indicating that type I hypersensitivity intermediated by IgE was involved in AFS.

[The countermeasure to pollinosis by a web site of Internet].

For the countermeasure to pollinosis, we opened "the web site of pollinosis by allergic group of otorhinolaryngology, Jikei Medical School" and provided the information of pollinosis for patients in the web site of internet from the spring of 1997. In the web site we kept to be informed of the pollen forecast, daily dispersed pollens, and medical information being renewed frequently of prevention and therapy for pollinosis. For the principle of therapy, we adopted the guideline for allergic rhinitis which was produced by Japan Allergic Societies and recommended visitors to get standard therapy for pollinosis. Consequently, the web site was accessed up to 160,000 times by the summer of 1999 and we received 204 medical questions by e-mail and answered to these all mails. We then made a questionnaire study after 3 each pollen seasons and received over 200 answers which showed that our fresh information was useful to decrease symptoms of pollinosis. These results show that information by web site seems to be useful for the countermeasure to pollinosis and will be more important to support medical treatment in hospitals in future.

Aerosolized Syk antisense suppresses Syk expression, mediator release from macrophages, and pulmonary inflammation.

Syk protein tyrosine kinase (PTK) is involved in signaling in leukocytes. In macrophages, Fcgamma-receptor cross-linking induces Syk PTK phosphorylation and activation, resulting in Syk-dependent events required for phagocytosis and mediator release. We hypothesized that Syk antisense oligodeoxynucleotides (ASO) delivered by aerosol to rat lungs in vivo would depress Syk PTK expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation. RT-PCR and RT-in situ PCR demonstrated that aerosolized Syk ASO administration reduced Syk mRNA expression from alveolar macrophages compared with cells isolated from sham-treated rats. Western blot analysis confirmed that Syk PTK expression was reduced after Syk ASO treatment. Compared with sham-treated rats (scrambled oligodeoxynucleotide), Syk ASO treatment suppressed Fcgamma-receptor-mediated nitric oxide (86.0 +/- 8.3%) and TNF (73.1 +/- 3.1%) production by alveolar macrophages stimulated with IgG-anti-IgG complexes. In contrast, Fcgamma-receptor-induced IL-1beta release was unaffected by Syk ASO treatment. Additionally, Syk ASO suppressed Ag-induced pulmonary inflammation, suggesting that Syk ASO may prove useful as an anti-inflammatory therapy in disorders such as asthma.

Reverse transcriptase in situ polymerase chain reaction for gene expression in rat mast cells and macrophages.

Direct reverse transcriptase in situ polymerase chain reaction (RT-in situ PCR) of selected mRNA expression in rat mast cells (MC) and alveolar macrophages (AM) was optimized. Rat peritoneal mast cells (PMC), rat cultured mast cells (RCMC), rat bronchoalveolar lavage cells (BALC) or rat cultured alveolar macrophages (NR8383) were studied for the detection of mRNA for beta-actin, TNF-alpha and/or CD8alpha. Each type of cell has unique optimal conditions for RT-in situ PCR. The following parameters were carefully evaluated for optimization: protease digestion, DNAse digestion, heparinase digestion, RT, PCR cycle number and signal development with chromagen. Heparinase digestion was required for PMC mRNA detection because they contain large amounts of heparin proteoglycan, which is a potent inhibitor of RT and Taq polymerase enzymes. Only a few PCR cycles were needed to produce a cytoplasmic signal for mRNA transcripts in RCMC, whereas other types of cells (PMC, BALC and NR8383) needed at least 20 cycles for mRNA detection. The mRNA signal in PMC was localized to the perinuclear region, whereas mRNA in other cell types (RCMC, BALC and NR8383) were detected throughout the cytoplasm. Furthermore, modified Southern blot analysis for TNF-alpha in RCMC treated with RT-in situ PCR demonstrated the specificity of amplification product. The modified and optimized protocols for this procedure were successfully applied to detect and localize several mRNA transcripts in rat MC and AM. The approach is valuable and can be used to further study selected gene expression in these and other cell types.

Novel CD8 molecule on macrophages and mast cells: expression, function and signaling.

BACKGROUND: We previously identified, using flow cytometry and in situ RT-PCR, a novel CD8 molecule on rat alveolar macrophages (AM) and mast cells (MC). RT-PCR also demonstrated that mouse AM express CD8 mRNA. Functional studies on rat AM determined that ligation of CD8 alpha- and beta-chains induced inducible nitric oxide synthase (iNOS) upregulation, nitric oxide (NO), TNF-alpha and IL-1beta (CD8alpha only) secretion. However, CD8 did not induce AM phagocytosis of IgG-opsonized or unopsonized particles. Rat MC stimulated through CD8 secreted NO and TNF-alpha, but not histamine. Because of its potential role in regulating cell function, we investigated the signaling pathways involved in macrophage CD8 stimulation. METHODS AND RESULTS: Inhibitor of src family kinases (PP1) significantly (p<0.05) inhibited CD8alpha (OX8 antibody)-induced iNOS upregulation, NO, TNF-alpha and IL-1beta production in rat AM. In addition, Ro 31-8220 (a PKC inhibitor) inhibited OX8-induced iNOS upregulation, NO and IL-1beta production, but did not inhibit TNF-alpha production. Using Syk antisense, we further determined that OX8 stimulation of NO is Syk kinase dependent. CONCLUSION: Studies on the signaling mechanisms of CD8 determined that src family kinases, PKC, and Syk kinase are involved in CD8 signaling. Additionally, CD8 may have differential signaling pathways, as an inhibitor to PKC downregulated OX8-induced IL-1beta, but not TNF-alpha release. Our studies demonstrate that AM CD8 is similar to T lymphocyte CD8 in that src kinases are involved in CD8-mediated signaling. However, p56(lck), which is expressed in T lymphocytes, has not been found in macrophages, suggesting that other src family kinases may be involved in AM and MC CD8 signaling.

Mast cells express novel CD8 molecules that selectively modulate mediator secretion.

CD8, a marker largely restricted to subsets of T lymphocytes and NK cells, was detected on freshly isolated rat peritoneal mast cells (PMC). Using flow cytometry, Percoll-enriched rat PMC (> or = 98% purity) were positive for the hinge region of CD8alpha (67.5 +/- 9.5%; Ab OX8) and CD8beta (27.8 +/- 2.3%; Ab 341). CD8+ PMC consisted of two populations, CD8alpha+ (22.5%) and CD8alpha+ beta+ (15.9%). Interestingly, G28, an Ab that identifies the IgV-like region of CD8alpha on T lymphocytes, did not bind PMC, suggesting that PMC CD8alpha is distinct from that on T lymphocytes. Moreover, a similar pattern of Ab positivity for CD8 was observed on a rat mast cell line, RBL 2H3. The presence of CD8alpha immunoreactivity on rat PMC was further confirmed by confocal microscopy. In situ reverse-transcription PCR and reverse-transcription PCR analysis demonstrated that PMC contained mRNA transcripts encoding CD8alpha. In functional studies of CD8 on PMC, both TNF-alpha and nitric oxide production were induced by OX8 (CD8alpha) and 341 Ab (CD8beta) in a dose-dependent manner. However, neither OX8 nor 341 induced histamine secretion from PMC. Ag-induced secretion of TNF-alpha, nitric oxide, and histamine was not affected by OX8 or 341 Abs, suggesting that there are distinct signaling mechanisms mediated by CD8 and Fc epsilonRI. These results indicate that rat PMC express functional CD8 molecules that may be distinct from those of T lymphocytes. The difference suggests there is a ligand other than MHC class I for mast cell CD8.

IL-4 and IL-6 production of bone marrow-derived mast cells is enhanced by treatment with environmental pollutants.

To examine the potential effects of environmental pollutants on the production of cytokines in mast cells, mouse bone marrow-derived mast cells (BMMC) were cultured at various concentrations of diesel exhaust particulates (DEP) or formaldeyhde. Proliferation of BMMC at 0.8, 2 and 4 microg/ml of DEP and 0.5 and 1 microg/ml of formaldehyde did not differ significantly from that of the controls (0 microg/ml) after 72 h culture, with the exception of a significant decrease in proliferation at 5 microg/ml of formaldehyde. Treatment with DEP or formaldehyde alone did not induce interleukin-4 (IL-4) or IL-6 production by BMMC. IL-4 and IL-6 production in BMMC stimulated with A23187 was higher in BMMC treated with low concentrations of DEP than in controls, but no increase was seen in BMMC treated with high DEP. IL-4 and IL-6 production in A23187-stimulated BMMC was significantly increased at 0.5 and 1 microg/ml formaldehyde but decreased at 5 microg/ml formaldehyde. After pretreatment with low DEP or formaldehyde alone for 24h, IL-4 production of BMMC stimulated with A23187 was lower in BMMC treated with low DEP or formaldehyde than in controls. Antigen-induced IL-4 production significantly increased in BMMC treated with 0.4 or 0.8 microg/ml DEP or 0.5 microg/ml formaldehyde, but antigen-induced IL-6 production in BMMC did not increase at low DEP or formaldehyde. Although the enhancement of IL-4 production of BMMC stimulated with A23187 plus DEP was not completely inhibited by 5x10(-4) M 2-mercaptoethanol (2-ME), treatment with 10(-7) M dexamethasone inhibited further IL-4 production. Cytokine production of mast cells is thus shown here for the first time to be modulated by treatment with DEP or formaldehyde. Environmental pollutants such as DEP and formaldehyde may thus affect the immune response via the modulation of cytokine production in mast cells.

[Relationship between the morphological changes of Japanese cedar (Cryptomeria japonica) pollen grains and the release of major allergens from the pollen].

Release of major allergens (Cry j 1, Cry j 2) from Japanese cedar (C. japonica) pollen grains treated with high pressure was investigated. C. japonica pollen grains crushed by high pressure treatment using FRENCH Pressure Cell Press released greater amounts of major allergens, particularly Cry j 2, compared to those without crush. This suggests that the cytoplasm of C. japonica pollen grains contains more Cry j 2 than previously reported. The effect of nasal fluid on the release of major allergens from C. japonica pollen grains was analyzed in vitro. Nasal fluid from patients with nasal allergy remarkably increased the release of major allergens from pollen grains, compared to controls, and the amount of Cry j 1 was greater than Cry j 2. Further studies revealed that nasal fluid affects the outer wall of pollen grains, where Cry j 1 is located, to a greater extent than its effect on the cytoplasm, where Cry j 2 is located.

[Evaluation of efficacy for Japanese cedar pollen-specific immunotherapy by lymphocyte proliferation test].

Peripheral mononuclear cells from 47 patients suffering from Japanese cedar pollinosis were stimulated with crude pollen extract of Cryptomeria Japonica (CJ) to evaluate the effect of CJ-specific immunotherapy. The stimulation index (SI) with crude pollen extract of CJ in the patients with excellent and good clinical outcome was significantly lower than those with poor outcome. The SI increased with the increasing symptoms in a dose dependent manner. In the patients under immunotherapy for long period, the good clinical outcome and low SI were gained. These results suggest that peripheral lymphocyte proliferation test should be a good objective indicator for allergen-specific immunotherapy.

[Comparative study on the assay for IgE antibodies specific for Chamaecyparis obtusa pollen between AlaSTAT and CAP-RAST].

Titers of IgE antibody specific for the pollen of Chamaecyparis obtusa (C. obtusa) were determined by AlaSTAT and CAP-RAST in 221 patients with Japanese cedar pollinosis. IgE antibody to C. obtusa tested positive by CAP-RAST at a higher rate (80.5%) than by AlaSTAT (52.6%). The results obtained from the two assays were compared with those from intradermal skin test. CAP-RAST had a higher sensitivity than that of AlaSTAT. Because the two methods showed no differences in the determination of IgE antibody specific for Cryptomeria japonica, the above differences between AlaSTAT and CAP-RAST are surmised to be ascribable to the differences of C. obtusa antigen used in the both assays.

Intranasal instillation of diesel exhaust particulates and antigen in mice modulated cytokine productions in cervical lymph node cells.

To investigate cytokine production stimulated by diesel exhaust particulates (DEP) and antigen through the intranasal route, mice were administered with DEP mixed with ovalbumin (OA) 3 times at an interval of 3 weeks. After the last instillation, cervical lymph node cells (LNC) were cultured in vitro with OA and antigen-presenting cells. The proliferative response to OA in cervical LNC from mice instilled with DEP and OA was noted to have increased significantly compared to mice instilled with OA alone. Interleukin 4 (IL-4) and interferon (IFN)-gamma in culture supernatants were measured with ELISA. OA-stimulated IL-4 production in cervical LNC from mice instilled with DEP and OA markedly increased beyond that in the control mice. In contrast, OA-stimulated IFN-gamma production in cervical LNC from mice instilled with OA was 3 times that for DEP and OA-instilled mice. OA-specific IgE antibody in sera showed a trend to be increased in mice intranasally instilled with DEP and OA. These results suggest that intranasal instillation of DEP and antigen in mice may modulate in vitro antigen-stimulated cytokine production from cervical LNC with a consequent increase in IgE antibody production.

Ganglioside GM3 inhibits interleukin-3-dependent bone marrow-derived mast cell proliferation.

To investigate the modulated proliferation of an interleukin-3(IL-3)-dependent cell by exogenous ganglioside GM3, mouse bone marrow-derived mast cells (BMMC) were cultured with various concentrations of GM3 in the presence of IL-3. By 4 weeks of culture, most of the nonadherent cells were alcian blue-positive mast cells. Culturing 2-week-cultured BMMC with GM3 for 1 week reduced the number of alcian blue-positive cells, but the increased total histamine content of BMMC was observed. To examine the effect of GM3 on the synergistic response by IL-3 and interleukin-4 (IL-4), 3-week-cultured BMMC were cultured with GM3 in the presence of IL-3 and IL-4 for 1 week. Although the addition of IL-4 to culture medium increased the number of BMMC, treatment with GM3 reduced its proliferative activity. Concerning the effect of GM3 on cell membrane, there are no changes in the expression of IgE receptors on BMMC treated with GM3 though a low concentration of GM3 increased it. However, the production of tumor necrosis factor-alpha from BMMC treated with GM3 was significantly suppressed. These results indicate that in vitro treatment with exogenous GM3 inhibited the proliferative response of IL-3-dependent mast cell populations and modulated its characteristics.

IL-4 production in mediastinal lymph node cells in mice intratracheally instilled with diesel exhaust particulates and antigen.

To clarify the relationship between air pollutants and IgE antibody production, interleukin 4 (IL-4) production was investigated in BALB/c mice intratracheally injected with diesel exhaust particulates (DEP) mixed with antigen (Ovalbumin (OA) or Japanese Cedar Pollen (JCP)). BALB/c mice were injected with DEP plus OA or OA alone three times with a 3-week interval. After the last instillation, proliferative response and lymphokine-producing activity of mediastinal lymph node cells (LNC) were examined in vitro. Proliferative response to OA in mediastinal LNC from mice injected with DEP plus OA was enhanced 4-17 times of that from control mice. IL-4-producing activity by OA stimulation also enhanced in mediastinal LNC from mice injected with DEP plus OA. A significantly larger amount of anti-OA IgE antibody was detected in sera from DEP- and OA-injected mice compared with those from control mice. The levels of IL-4, estimated by JCP antigen in mediastinal LNC, from mice injected with DEP plus JCP were two-fold higher than those from mice injected with JCP alone. These results suggest that intratracheal instillation of DEP affects antigen-specific IgE antibody responses via local T-cell activation, especially enhanced IL-4 production.