Kenji Kawada

Immunoglobulin E production in mice by means of contact sensitization with a simple chemical, hapten

Production of IgE caused by repeated topical application of 2,4-dinitrofluorobenzene (DNFB) to the ears of BALB/c mice was investigated. Ear thickness increased in proportion to the number of applications. Eczematous changes of the skin included marked infiltration of neutrophils, eosinophils, and monocytes and hypertrophy of the epidermis. Ear thickness due to inflammation reached a maximum 24 hours after the second, third, fourth, and fifth applications. The strong expression of interferon-gamma and IL-2 messenger RNA (mRNA) in the skin lesions indicated the participation of Th1 type helper T cells in the delayed-type hypersensitivity reaction. After the fifth application, the mice showed an immediate cutaneous reaction in addition to the delayed-type hypersensitivity reaction. The immediate reaction appeared within 1 hour of application. Hapten-specific IgE also was detected in serum from the mice, and the expression of germline and productive Cepsilon mRNA was detected in cervical lymph nodes, whereas productive Cepsilon mRNA was detected in the spleen. These results indicate that five topical applications of DNFB to the ear produce local eczematous dermatitis and increase serum hapten-specific IgE level in mice. Eczematous dermatitis is mainly caused by Th1 type T cells, and IgE production is mediated by Th2 type T cells in the cervical lymph nodes. In addition, IgE class switching occurs in the cervical nodes and IgE production occurs in both lymph nodes and spleen.

Different role of IL‐4 in the onset of hapten‐induced contact hypersensitivity in BALB/c and C57BL/6 mice

To study the role of interleukin (IL)‐4 in the onset of contact hypersensitivity (CH) in mice, the effect of IL‐4 gene‐depletion and anti‐IL‐4 monoclonal antibody treatment on dinitrofluorobenzene (DNFB)‐induced CH was examined. Simultaneously, to clarify the effect of background gene, DNFB‐induced CH in BALB/c and C57BL/6 mice was compared. Five repeated topical applications of DNFB to the ears of mice resulted in CH of the ears in terms of increases in ear thickness and histopathological changes. The magnitude of ear thickness increase in BALB/c mice was almost three times greater than that in C57BL/6 mice. The CH in BALB/c mice was significantly suppressed by IL‐4 gene‐depletion and anti‐IL‐4 monoclonal antibody treatment. In contrast, the symptoms of dermatitis in C57BL/6 mice were slightly affected by the same treatment. These changes corresponded well to the production of specific IgE antibody. Total IgE antibody production and the expression of productive Cε mRNA were dramatically suppressed by IL‐4 gene‐depletion and anti‐IL‐4 treatment in BALB/c and C57BL/6 mice. Neither total IgG nor IgM levels in either strain of mice was altered by depletion of IL‐4. The expression of IFN‐γ in the skin lesion was dramatically suppressed by IL‐4 gene‐depletion in BALB/c mice, but not in C57BL/6 mice. These findings indicate that IL‐4 plays an important role in the onset of DNFB‐induced CH in BALB/c mice, but not in C57BL/6 mice.

Adhesion and Colonization of Enterohemorrhagic Escherichia coli O157:H7 in Cecum of Mice

Infectious diseases due to enterohemorrhagic Escherichia coli (EHEC) are characterized by diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. The adherence of EHEC on intestinal epithelial cells is a first step for developing these diseases. In the present study, we examined whether EHEC O157: H7 adhere to intestinal epithelial cells of mice and cause F‐actin accumulation in the epithelial cells following the intragastric inoculation of the pathogen. Fecal shedding of the EHEC O157:H7 strain was observed in ICR mice up to 3 weeks. Fecal shedding periods of the type III secretion system‐related gene (espA and sepL) deletion mutants were clearly shorter than that of the wild‐type EHEC O157:H7 strain. The EHEC O157:H7 colonies were found on the epithelial surfaces of the ceca in association with F‐actin accumulation beneath the attached bacteria.

Cyclosporin A and FK-506 Inhibit Development of Superantigen-potentiated Collagen-induced Arthritis in Mice.

1. Staphylococcal enterotoxine B (SEB; superantigen) accelerated the onset of arthritis in mice preimmunized with type II collagen (SEB-potentiated collagen-induced arthritis). Cyclosporin A and FK-506 inhibited the induction and development of clinical signs and histopathological changes of SEB-potentiated collagen-induced arthritis in mice. 2. Simultaneously, both cyclosporin A and FK-506 inhibited the development of humoral and cellular immunity to type II collagen. 3. The expression of IL-2 receptor (CD25) by SEB on splenocyte T cells from collagen-preimmunized mice was inhibited by both agents in ex vivo experimentation.

Effect of KE-298 on Experimental Arthritis in Mice

KE-298 is a new immunomodulatory agent with a chemical structure similar to that of D-penicillamine. We compared the effects of KE-298 on type II collagen-induced arthritis (CIA) in mice with those of prednisolone. KE-298 at a dose of 25 mg/kg showed only a decrease in the progression of foot pad swelling. At doses of 50 and 100 mg/kg, however, KE-298 inhibited the severity and development of the collagen-induced arthritis index, the progression of foot pad swelling, bone damage and histopathological changes. These inhibitory effects were more pronounced at the dose of 50 mg/kg than at 100 mg/kg. KE-298 also significantly inhibited the delayed-type hypersensitivity (DTH) response to type II collagen, but did not affect the production of anti-type II collagen IgG antibody in arthritic mice. To determine the inhibitory mechanism of KE-298, we studied the effect of KE-298 on IL-1 beta and TNF-alpha production in mice. We found that KE-298 inhibited bacterial lipopolysaccharide (LPS)-induced IL-1 beta production at doses of 50 and 100 mg/kg. It inhibited the production of TNF-alpha at the dose of 50 mg/kg, but not at 100 mg/kg. In summary, at appropriate dosages, KE-298 inhibited CIA and TNF-alpha production in mice. KE-298 also inhibited the DTH reaction to type II collagen and LPS-induced IL-1 beta production in a dose-related fashion. These findings suggest that these effects of KE-298 are closely related to its immunomodulatory action.

Morphological studies on nephrotoxic serum nephritis accelerated with rabbit IgG in mice.

A glomerulonephritis was induced in mice by injection of subnephrotoxic dose of nephrotoxic serum (NTS) after preimmunization with rabbit IgG. In order to characterize this glomerulonephritis, light, electron and immunofluorescence microscopic studies were carried out 15 days after NTS injection, the time when increases in urinary protein and serum cholesterol and a decrease in serum albumin were apparent.Characteristic changes were widespread thickening of capillary walls and narrowing of the capillary lumen owing to widening of mesangial areas. In those capillary walls, the mesangial interposition into subendothelial areas was often noted ultrastructurally and double track was confirmed on sections stained with PAM. Linear deposition of mouse IgG was detected in capillary walls by immunofluorescence. In severely affected glomeruli, PAS-positive hyaline nodular lesion was observed light microscopically and massive mesangial deposits ultrastructurally.Visceral epithelial cells demonstrated fusion of the foot processes, microvilli formation, occasional proliferation and enlargement. Parietal epithelial cells proliferated, forming cellular or fibrocellular crescent.Based on these characteristics, it appears this nephritic model shares a common pathology with human membranoproliferative glomerulonephritis type 1 and crescentic glomerulonephritis and can be considered an appropriate model for producing severe nephritis for short periods.