Kazuki Hirahara

Oral administration of a dominant T-cell determinant peptide inhibits allergen-specific TH1 and TH2 cell responses in Cry j 2–primed mice

BACKGROUND Oral immunotherapy with a peptide for allergic immune responses is theoretically a promising therapy but has not been established yet. OBJECTIVE To evaluate immune suppressive efficacy of oral administration of an immunodominant peptide, we investigated changes in T-cell proliferation, TH1 - and TH2 -cytokine production, and TH1 - and TH2 -mediated antibody production in mice after oral administration of a peptide. METHODS Peptide p246-259, containing a dominant T-cell determinant of Cry j 2, which is the major allergen in Japanese cedar pollen, was used in this study. Groups of mice received p246-259 or PBS alone before or after they were primed intranasally with Cry j 2 and cholera toxin. In another experiment mice were primed intraperitoneally with Cry j 2 and alum. Proliferative response and cytokine production by nasal-associated lymph node cells against Cry j 2 were investigated. Amounts of systemic anti-Cry j 2 IgE and IgG antibodies were also measured. RESULTS Oral administration of the peptide to mice before, or even after, the sensitization induced oral tolerance in T-cell responses against the allergen; the tolerance was associated with decreased production of TH1 (IFN-gamma and IL-2) and TH2 (IL-4) cytokines. Allergen-specific TH1 -mediated (IgG2a and IgG2b) and TH2 -mediated (IgG1 and IgE) antibody responses were also inhibited. CONCLUSIONS Oral administration of a dominant T-cell determinant peptide induces immunologic tolerance in both TH1 and TH2 cell responses against the whole protein allergen. Our study is the first, to our knowledge, to demonstrate the potential for peptide-based oral immunotherapy in order to treat allergic immune responses.

Identification of the fused bicyclic 4-amino-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors

2-Phenyl-4-piperidinyl-6,7-dihydrothieno[3,4-d]pyrimidine derivative (2) was found to be a new PDE4 inhibitor with moderate PDE4B activity (IC50=150 nM). A number of derivatives with a variety of 4-amino substituents and fused bicyclic pyrimidines were synthesized. Among these, 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative (18) showed potent PDE4B inhibitory activity (IC50=25 nM). Finally, N-propylacetamide derivative (31b) was determined as a potent inhibitor for both PDE4B (IC50=7.5 nM) and TNF-α production in mouse splenocytes (IC50=9.8 nM) and showed good in vivo anti-inflammatory activity in the LPS-induced lung inflammation model in mice (ID50=18 mg/kg). The binding mode of the new inhibitor (31e) in the catalytic site of PDE4B is presented based on an X-ray crystal structure of the ligand-enzyme complex.

Three T‐cell determinants of Cry j 1 and Cry j 2, the major Japanese cedar pollen antigens, retain their immunogenicity and tolerogenicity in a linked peptide

It has been demonstrated in detail that administration of a dominant T‐cell determinant to animals induces activation or immunological tolerance of T cells. However, it has not been determined whether multiple T‐cell determinants, when integrated into a single peptide, retain their potential to induce T‐cell activation and tolerance. We prepared a synthetic peptide comprising three T‐cell determinants of Cry j 1 and Cry j 2, the major Japanese cedar pollen antigens, and investigated the immunogenicity and tolerogenicity of each T‐cell determinant in the linked peptide by means of lymph node cell proliferation assays using mice. Lymph node cells from mice immunized with each of the three T‐cell determinants proliferated against the linked peptide in a dose‐dependent manner, similar to that of the immunized peptide. Lymph node cells from mice immunized with the linked peptide proliferated against all of the three T‐cell determinants. In addition, the degree of proliferation against the three T‐cell determinants occurred according to their original immunogenicity, as observed in the native protein antigens. Oral administration of the linked peptide to mice before they were immunized with Cry j 1 and Cry j 2 inhibited lymph node cell proliferation against the three T‐cell determinants, depending on the dose of the linked peptide administered. In conclusion, it was demonstrated that three T‐cell determinants retain their original immunogenicity and tolerogenicity in a linked peptide comprising them.

Novel CC chemokine receptor 4 antagonist RS-1154 inhibits ovalbumin-induced ear swelling in mice

CC chemokine ligand 17 (CCL17/thymus and activation-regulated chemokine: TARC) and CCL22 (macrophage-derived chemokine: MDC) selectively bind to CC chemokine receptor 4 (CCR4). The CCR4 system is considered to be responsible for the pathology of allergic diseases such as atopic dermatitis. To find and develop potential medicines against allergic diseases, we screened an in-house library to search for compounds having a profile as a CCR4 antagonist. From among the screening hits, we focused on 3-{2-[(2R)-2-phenyl-4-(4-pyridin-4-ylbenzyl)morpholin-2-yl]ethyl}quinazoline-2,4(1H,3H)-dione (named RS-1154), which had been newly synthesized in our laboratory. This compound inhibited the binding of [(125)I]CCL17 to human CCR4-expressing CHO cells with an IC(50) value of 27.7 nM and moreover inhibited CCL17-induced migration of DO11.10 mice-derived T helper 2 cells with an IC(50) value of 1.5 nM in vitro. We then examined the effect of RS-1154 in an ovalbumin-induced ear swelling assay. The ear thickness was decreased by intravenous administration of anti-CCL17 or anti-CCL22 antibodies, suggesting that the CCR4 system is involved in the ear swelling. Though partially, the oral administration of RS-1154 also significantly ameliorated the ear swelling at the doses of 30 and 100 mg/kg. Furthermore, the serum level of interleukin-4 decreased after the administration of RS-1154. In this study, we succeeded in obtaining a newly-synthesized compound, RS-1154, which has a potential to inhibit the chemotaxis of T helper 2 cells in vitro and to ameliorate ovalbumin-induced ear swelling in vivo. These results raise the possibility that RS-1154 or one of derivatives might become a therapeutic agent for atopic dermatitis patients.

Inhibition of neutrophil migration in mice by mouse formyl peptide receptors 1 and 2 dual agonist: indication of cross-desensitization in vivo.

It has been reported that the stimulation of neutrophils with N‐formyl‐Met‐Leu‐Phe (fMLF), an agonist for formyl peptide receptor (Fpr) 1, renders cells unresponsive to other chemoattractants in vitro. This is known as cross‐desensitization, but its functional relevance in neutrophil migration in vivo has not been investigated. Here, we show that precedent stimulation of mouse neutrophils with compound 43, a non‐peptidyl agonist for mouse Fpr1 and Fpr2, rendered the cells unresponsive to a second stimulation with C5a, leukotriene B4, or keratinocyte‐derived cytokine (KC) in calcium mobilization and chemotaxis assays in vitro. The expression of chemokine (C‐X‐C motif) receptor 2 (CXCR2) on the surface of neutrophils was concomitantly diminished by stimulating the cells with the compound. Moreover, oral administration of the compound to mice before they were exposed to lipopolysaccharide (LPS) aerosol resulted in a dose‐dependent reduction in the neutrophil count in bronchoalveolar lavage fluid. The expression of CXCR2 on blood neutrophils was also reduced in the compound‐administered mice. The recipient mice that underwent adoptive transfer of fluorescence‐labelled neutrophils that had been incubated with the compound showed a substantial decrease in neutrophil counts in bronchoalveolar lavage fluid after they were exposed to LPS, when compared with the control mice to which vehicle‐treated neutrophils had been transferred. These results are consistent with the idea that the agonist for Fpr1 and Fpr2 induced cross‐desensitization in neutrophils and attenuated neutrophil migration into the airways. Our results also revealed the unpredicted effect of an Fpr1 and Fpr2 dual agonist, which may act as a functional antagonist for multiple chemoattractant receptors in vivo.

Synthesis and biological evaluation of 5-carbamoyl-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors.

5-Carbamoyl-2-phenylpyrimidine derivative 2 has been identified as a phosphodiesterase 4 (PDE4) inhibitor with moderate PDE4B inhibitory activity (IC50=200 nM). Modification of the carboxylic acid moiety of 2 gave N-neopentylacetamide derivative 10f, which had high in vitro PDE4B inhibitory activity (IC50=8.3 nM) and in vivo efficacy against lipopolysaccharide (LPS)-induced pulmonary neutrophilia in mice (ID50=16 mg/kg, ip). Furthermore, based on the X-ray crystallography of 10f bound to the human PDE4B catalytic domain, we designed 7,8-dihydro-6H-pyrido[4,3-d]pyrimidin-5-one derivative 39 which has a fused bicyclic lactam scaffold. Compound 39 exhibited excellent inhibitory activity against LPS-induced tumor necrosis factor alpha (TNF-α) production in mouse splenocytes (IC50=0.21 nM) and in vivo anti-inflammatory activity against LPS-induced pulmonary neutrophilia in mice (41% inhibition at a dose of 1.0 mg/kg, i.t.).

Approaches to immunotherapies for Japanese cedar pollinosis

Japanese cedar (Cryptomeria japonica; CJ) pollinosis is a typical type I allergy induced by CJ pollen and one of the most common allergic diseases in Japan. New immunotherapies have been developed for treatment of CJ pollinosis. We focus here on new immunotherapies for CJ pollinosis including sublingual immunotherapy with crude extract of CJ antigen, oral immunotherapy with transgenic rice expressing CJ allergens, a peptide vaccine using T cell epitopes of CJ allergens, DNA vaccines encoding either the CJ allergen gene or T cell epitope gene, and adjuvant-conjugated vaccines using CJ allergen conjugated with adjuvants such as CpG oligodeoxynucleotide or pullulan.

Melanocortin receptors 1 and 5 might mediate inhibitory effects of α-melanocyte-stimulating hormone on antigen-induced chronic allergic skin inflammation in IgE transgenic mice.

TO THE EDITOR Atopic dermatitis (AD) is a chronic allergic disease in skin with severe irritation and inflammation, and most patients with AD show elevated plasma levels of IgE against large numbers of allergens. It is known that IgE is involved in type I allergy, which is an immediate hypersensitivity through mast cells with IgE. However, it is not clear how IgE affects chronic allergic diseases such as AD. Anti-IgE antibody (omalizumab) has been shown to improve symptoms in not only allergic rhinitis, which is a typical type I allergy disease, but also AD and chronic asthma, which are chronic allergic diseases (Sheinkopf et al., 2008). We focused on elucidating the mechanism for IgE-mediated chronic inflammation to find and establish a original treatment for chronic allergic diseases. In 2,4,6-trinitrophenol-specific IgE transgenic mice (TNP-IgE Tg mice), a single subcutaneous injection of a multivalent antigen such as TNP-conjugated ovalbumin (TNP11-OVA) to the ear induces tri-phase ear swelling (Matsuoka et al., 1999; Sato et al., 2003). The immediate-phase (within 1 hour after antigen challenge) and late-phase (within 6–10 hours after antigen challenge) ear swelling are typical responses of type I allergy, whereas the thirdphase ear swelling (began 2 days later and peaked on days 3–4 after antigen challenge) especially reflects chronic IgE-mediated severe inflammation, which is induced by the multivalent antigen (Sato et al., 2003). In the histological change of the third-phase ear swelling, massive infiltration of inflammatory cells such as eosinophils, neutrophils, and hyperplastic epidermis with hyperkeratosis were observed. Anti-histamine drugs did not show any significant effect on the third-phase swelling, whereas Cyclosporine A inhibited swelling and cellular infiltration (Sato et al., 2003). Moreover, we showed that a topical steroid strongly inhibited swelling and cellular infiltration (Supplementary Figure S1 online). The third-phase ear swelling in TNP-IgE Tg mice has hallmarks of chronic allergic inflammation in AD at both points of IgE-mediated intense chronic inflammation and effects of therapeutic drugs. To identify molecules that are responsible for IgE-mediated chronic inflammation, we investigated the gene expression profiles of antigenadministered ears in TNP-IgE Tg mice by Affymetrix GeneChip analysis (Santa Clara, CA). The thickness of TNP11OVA-administered ears 24 hours after antigen challenge (just before the thirdphase ear swelling) has the same degree as that of OVA-administered (control)

The incidence of Japanese cedar pollinosis and sensitization to the pollen allergens among Japanese monkeys in a troop

The natural occurrence of Japanese cedar (Cryptomeria japonica; CJ) pollinosis has been reported in Japanese monkeys (Macaca fuscata), an appropriate animal model for developing antipollinosis therapies. However, there has been no study on the incidence of Japanese cedar pollinosis in monkeys. To evaluate the incidence of CJ pollinosis in Japanese monkeys, we investigated the presence of pollinosis symptoms among monkeys in a troop, and the response to CJ allergens in pollinosis monkeys. We examined the presence of pollinosis symptoms in 272 monkeys in a troop throughout the CJ pollination season (February to April). Of the 272 monkeys, 21 (7·7%) showed pollinosis symptoms during the CJ pollen season. Blood samples were taken from the 21 monkeys that showed pollinosis symptoms and were tested for the presence of immunoglobulin E (IgE) antibody for CJ allergens. All 21 monkeys with CJ pollinosis had anti‐CJ IgE. Of the 21 monkeys, peripheral blood mononuclear cells (PBMC) could be taken from 12, all of which showed CJ allergen‐specific PBMC proliferation. The incidence of CJ pollinosis in a troop was 7·7%. The monkeys with CJ pollinosis demonstrated specific IgE and PBMC proliferation for CJ allergens.

Elucidation of Distinct Roles of Guinea Pig CXCR1 and CXCR2 in Neutrophil Migration toward IL-8 and GROα by Specific Antibodies

Chemokine receptors CXCR1 and CXCR2 are conserved between guinea pigs and humans, but the distinct role of each receptor in chemotactic responses of neutrophils against chemokine ligands has not been elucidated due in part to the lack of specific inhibitors against these receptors in guinea pigs. In this study, we investigated the roles of guinea pig CXCR1 and CXCR2 on neutrophils in chemotactic responses to guinea pig interleukin (IL)-8 and growth-regulated oncogene (GRO)α by using specific inhibitory antibodies against these receptors. Neutrophil migration induced by IL-8 was partially inhibited by either anti-CXCR1 antibody or anti-CXCR2 antibody. In addition, the migration was inhibited completely when both anti-CXCR1 and anti-CXCR2 antibodies were combined. On the other hand, neutrophil migration induced by GROα was not inhibited by anti-CXCR1 antibody while inhibited profoundly by anti-CXCR2 antibody. These results indicated that CXCR1 and CXCR2 mediated migration induced by the IL-8 synergistically and only CXCR2 mediated migration induced by GROα in guinea pig neutrophils. Our findings on ligand selectivity of CXCR1 and CXCR2 in guinea pigs are consistent with those in humans.