Uki Yamashita

Interleukin-4 as a potent inhibitor of bone resorption

A possible role of interleukin-4 (IL-4) in the regulation of bone turnover was assessed by employing a 45Ca prelabeled-fetal mouse long bone culture system. IL-4 inhibited the bone resorption stimulated by parathyroid hormone (PTH), PTH related protein (PTHrP), 1 alpha, 25, dihydroxy-vitamin D3 [1 alpha, 25 (OH)2 D3], interleukin-1 alpha and - 1 beta (IL-1 alpha, IL-1 beta) and prostaglandin E2 (PGE2). Anti-IL-4 on monoclonal antibody abolished the inhibitory effect of IL-4 on the bone resorption. These results suggest that IL-4 may play an important role on the inhibitory regulation of bone resorption.

Biphasic changes in behavioral, endocrine, and sympathetic systems in adjuvant arthritis in Lewis rats

Adjuvant arthritis (AA) is an experimental model for rheumatoid arthritis, and is induced most easily in inbred Lewis rats by an intradermal injection of heat-killed Mycobacterium tuberculosis (MT) in incomplete Freunds adjuvant. Susceptivity to the arthritis in Lewis rats is thought to be related to a defect in their responses of the hypothalamo-pituitary-adrenal (HPA) axis to the disease. Because the use of an inbred strain is necessary for our immunological studies, we examined in Lewis rats changes in behavior, the HPA axis, and sympathetic nerve activities during development of the adjuvant arthritis. Following intradermal injections of heat-killed MT in adjuvant, the arthritis began to develop on day 12, reaching its maximum severity on day 21, and remained at the level for over a month. The body temperature rose from day 0 to 5 (the primary phase--before the onset of the arthritis). It then fell to normal temperature, and again rose from day 10 to 21 (the secondary phase--with fully developed arthritis). The behavioral (physical activity, food, and water intake) and hormonal parameters [plasma adrenocorticotropic hormone (ACTH) and corticosterone levels] also changed in two phases, similar to those observed in the temperature responses. No change in plasma vasopressin level was observed. Sympathetic nerve activities, assessed by changes in plasma noradrenalin levels, increased more in the primary than in the secondary phase. The possible causes for the biphasic changes associated with development of arthritis are discussed.

Effect of hyperbaric oxygenation on macrophage function in mice

Hyperbaric oxygenation (HBO) has an immunosuppressive effect. The possible mechanisms of this immunosuppressive effect were assessed by determining the production of interleukin-1 (IL-1), interleukin-6 (IL-6), and prostaglandin E2 (PGE2), as well as phagocytosis by splenic macrophages from HBO-treated mice. Although HBO treatment did not have any significant effect on IL-6 production and phagocytotic activity, a marked decrease in IL-1 production and a significant decrease in PGE2 production were observed. These results suggest that the reduction of IL-1 production may play an important role in the immunosuppressive effect of HBO.

Suppressive effect of hyperbaric oxygenation on immune responses of normal and autoimmune mice

We studied the effect of hyperbaric oxygenation (HBO) on immune responses in normal and autoimmune mice. Mice were exposed to HBO in an animal chamber at a pressure of 252.5 kPa for 1 h and once a day for 5 days. The immunization of C3H/He mice with sheep erythrocytes induced marked anti‐sheep erythrocyte antibody response on day 7. However, this response was markedly suppressed in HBO‐treated mice. The suppression is dependent on the duration of HBO and it works on the early and the late stage of antibody responses. HBO suppresses the development of both sheep erythrocyte‐specific B cells and helper T cells after the immunization. Then, we tried to expose autoimmune mice to HBO. Spontaneous immunoglobulin production of NZB and MRL/Ipr spleen cells was also significantly suppressed by HBO. Furthermore, long term HBO exposure results in the suppression of the development of autoimmune symptoms such as proteinuria, facial erythema and lymphadenopathy in MRL/lpr mice. All these results suggest that HBO is applicable for the treatment of autoimmune diseases.

Selective depletion of immature thymocytes by oral administration of ethylene glycol monomethyl ether

Although immunotoxicity of ethylene glycol monomethyl ether (EGME) has been strongly suspected, functional evaluation of the immune response in EGME-treated animals was negative in previous studies. We observed a decrease in thymic cellularity and increases in the various ex vivo immunological assays in mice, orally administered with EGME 0.5 or 1.0 mg/g body weight daily for 5 or 10 days: ex vivo lymphoproliferative responses to concanavalin A, in vitro induction of trinitrophenyl (TNP)-specific cytotoxic T-cell activity of thymocytes and splenocytes. Histopathological examination of the thymus of the treated mice disclosed a markedly atrophic cortex and almost intact thymic medulla. Study of thymocyte surface markers revealed that CD4+/CD8+, Thy-1+, PNA+ immature thymocytes were relatively decreased in EGME-gavaged mice and that, thus, ratios of CD4-/CD8+, H2+ mature thymocytes were enriched. These findings indicate that oral administrations of EGME selectively deplete immature thymocytes in mice. Although the mechanism of action remains unknown, the EGME-induced immature thymocyte depletion is not considered to be due to lymphocidal action of corticosteroids.

Suppressive Activity of Interleukin 4 on the Induction of Antigen-specific Cytotoxic T Cells in Humans

The effect of interleukin 4 (IL 4) on the induction of cytotoxic T cells (CTL) was studied by using human peripheral blood lymphocytes in vitro. IL 4 suppressed the induction of CTL specific for allogeneic antigens in a concentration‐dependent manner. However, IL 4 did not suppress proliferative responses induced with allogeneic antigens or mitogens. The suppressive effect of IL 4 on CTL induction was observed when IL 4 was added at the early period of the CTL induction culture, but not at the later period. Furthermore, IL 4 did not suppress the effector function of CTL to target cells. IL 4 suppressed the production of IL 1 by monocytes/macrophages and the production of IL 2 and the expression of IL 2 receptors on T cells. Moreover, IL 4 suppressed the induction of lymphokine‐activated killer cells. These results suggest that IL 4 has a suppressive activity on the induction of killer cells in humans.

B cell mitogenic activity of house dust mite, Dermatophagoides farinae, antigens

The effect of mite antigens on murine and human lymphocytes was studied in vitro. Antigens prepared from Dermatophagoides farinae feces and bodies stimulated normal murine spleen cells to proliferate in a dose-dependent manner. The responder cells are B cells, because the response was reduced by the treatment of spleen cells with anti-immunoglobulin antibody and complement, but not with anti-Thy 1 antibody and complement. Furthermore, nylon column-purified T cells did not respond. The stimulation of B cells with mite antigens was not due to the contamination of lipopolysaccharide, a representative B cell mitogen, because C3H/HeJ spleen cells which are low responders to lipopolysaccharide could respond to mite antigens. These antigens induced not only proliferative response of murine B cells, but also immunoglobulin production. By gel-filtration column chromatography, the active fractions were eluted around the molecular weight of 150-155 kDa. Furthermore, mite antigens also stimulated human B cells to proliferate and to produce immunoglobulin. All these results suggest that mite antigens are a potent B cell mitogen and this activity might concern the induction of allergic reaction.

B Cell Mitogenic Activity of Sea Squirt Antigen

The activity of sea squirt antigen, one of the allergy-inducing substances for humans, on murine and human lymphocytes was studied in vitro. Sea squirt antigen stimulated normal mouse spleen cells to proliferate, as detected by [3H]-TdR incorporation, in a dose-dependent manner. The responder cells are B cells because the response was reduced by the treatment of spleen cells with anti-immunoglobulin antibody and complement and passing through a nylon wool column, but not with anti-Thy-1 antibody and complement. Spleen cells of C3H/HeJ mice, which are lipopolysaccharide low responders, were also stimulated as well as spleen cells of C3H/HeN mice, suggesting that this response is not due to lipopolysaccharide in the antigen fraction. Sea squirt antigen stimulated not only proliferative response of B cells, but also polyclonal immunoglobulin production. Furthermore, sea squirt antigen also stimulated human lymphocytes to proliferate and to produce immunoglobulin. All these results suggest that sea squirt antigen has mitogenic activity on B cells, and this ability is concerned with the induction of allergic reaction.

Identification of T-cell epitope sequences on an important mite antigen

Background T‐cell epitopes on Der 1 and Der 2 groups, the major mite allergens, have been intensively analysed, while those on the other important allergens remain to be elucidated. We have cloned four cDNAs coding for important mite allergens on the basis of frequency and capacity of IgE binding. Stimulatory action of glutathione S‐transferase‐fused Mag1 on lymphocytes from mite‐allergic patients was relatively high among them.

B‐B Cell Interactions in the Spontaneous Activation of B Cells in Autoimmune NZB Mice

We analyzed the mechanism of spontaneous B cell activation in lupus mice by using anticlass‐II antibody in vitro. The in vitro culture of B cells from old NZB mice markedly produced Ig without any stimulation, while B cells from NZW mice did not. The addition of anticlass‐II antibody (anti‐Iad antibody) to the culture inhibited Ig production of NZB B cells in a concentration‐dependent manner. On the other hand, the addition of anticlass‐I antibody (anti‐H‐2Dd antibody) and anticlass‐II antibody with different specificity (anti‐Iak) gave no effect on the Ig production of NZB B cells. When mitomycin C‐treated B cells were added to in vitro culture of responder B cells as a stimulator, Ig production of responder B cells was enhanced in a concentration‐dependent manner. However, the enhancing effect of the stimulator B cells was abrogated by the pretreatment with anticlass‐II antibody. The stimulator B‐cell activity to NZB B cells was marked in NZB B cells, moderate in NZB/W F1 B cells, and weak in NZW B cells. Furthermore, the stimulator B‐cell activity with regard to NZB B cells was marked in old female NZB B cells, moderate in old male NZB B cells, and weak in young NZB B cells. The expression of class II antigens on the surface of old female NZB B cells was significantly higher than that of old male NZB and young NZB B cells. These results suggest that in lupus mice the spontaneous B‐cell activation is induced by an abnormal B‐B cell interaction mediated by class II antigens.