Hideaki Iribe

Augmentation of the proliferative response of thymocytes to phytohemagglutinin by the muramyl dipeptide

A synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine or muramyl dipeptide (MDP) and adjuvant-active analogs, but not lipopolysaccharide (LPS), exhibited the augmenting effect on the proliferative response of thymocytes to phytohemagglutinin (PHA). MDP also had a comitogenic effect on PHA-stimulated T lymphocytes. It was shown that the thymocyte-stimulating effect of MDP is not through the production of the monikines by MDP-stimulated macrophages and that MDP has a direct action on lymphocytes.

Macrophage-stimulating effect of a synthetic muramyl dipeptide and its adjuvant-active and -inactive analogs for the production of T-cell activating monokines.

Abstract A synthetic muramyl dipeptide (MDP), which has the minimal adjuvant-active structure that can substitute for Mycobacterium in complete Freunds adjuvant, could stimulate peritoneal macrophages of the guinea pig to produce T-cell activating soluble factors. The culture supernatant of MDP-stimulated macrophages could help the proliferative response of the T-lymphocyte-enriched fraction of lymph nodes to PHA. It also helped the antigenic activation of immune T lymphocytes deprived of macrophages to produce macrophage migration inhibitory factor (MIF). Gel filtration of the culture supernatant showed that both of the helping activities were found mainly in the fraction emerging at the 53,000- to 85,000-MW range. A series of MDP analogs which are known to be adjuvant active or inactive was tested for their ability to stimulate macrophages to produce these factors. The adjuvant activity of these synthetic compounds was found to be parallel to the ability to stimulate the production of these T-cell activating monokines. Since both of these activities are highly dependent on the precise stereochemical structures of the MDP analogs, the parallelism of these activities would suggest the possibility that the immunopotentiating effect of the MDP and its analogs is related at least partially to their activity to stimulate macrophages to produce T-cell activating monokines.

Characterization of the antibody response against the type II collagen induced by anti-idiotypic antibody

We reported that rabbit anti-idiotypic antibody (Ab2) against mAb, termed 1-5 (Ab1) and reactive with human type II collagen (CII) induced antibody response to CII in DBA/1J mice susceptible to collagen-induced arthritis. In the present study, we further characterized the anti-CII antibody response elicited by Ab2 with respect to epitope specificity, putative genetic background, and IgG subclass. Most of anti-CII antibodies (polyclonal Ab3) derived from Ab2-immunized mice were of the IgG1 subclass. We purified polyclonal Ab3, using a CII-coupled immunoadsorbent column and we developed monoclonal Ab3 from Ab2-immunized mice. Both purified polyclonal Ab3 and two monoclonal Ab3s specifically reacted with a selected epitope on CII, recognized by Ab1. The anti-CII antibody response stimulated by Ab2 was observed in DBA/1J (H-2q, Igh-1c) and DBA/2 (H-2q, Igh-1c) mice, but not in the BALB/c (H-2d, Igh-1a) and C57BL/6 (H-2b, Igh-1b) strains, thereby suggesting that the anti-CII antibody response elicited by Ab2 is controlled by the Igh gene.

Biological Activities of Guinea Pig Mitogenic Factor from Immune Lymphocytes Stimulated with Antigen

Mitogenic factor was produced by sensitized guinea pig lymph node cells stimulated with a specific antigen. Both T lymphocytes and macrophages were required for the production of this factor. The culture supernatant of lymphocytes containing the mitogenic factor exhibited a strong helping effect on the proliferative response of T lymphocytes to phytohemagglutinin (PHA). Mitogenic factor and the factor with the helping activity coeluted in the molecular weight range of 25,000‐35,000 daltons in gel filtration. Furthermore the fraction containing mitogenic factor was found to support the proliferation of lymphoblasts induced by PHA or antigen, suggesting that the mitogenic factor may be the guinea pig equivalent of T cell growth factor (TCGF) reported in the mouse, rat, and human.

T cell line specific for bacterial peptidoglycan subunit: Possible role of the COOH-terminal amino acid of the disaccharide tetrapeptide in binding to the T cell receptor

The T cell line specific for a bacterial cell wall peptidoglycan subunit, disaccharide tetrapeptide of diaminopimelic acid type, was examined for epitope specificity in elicitation of delayed-type hypersensitivity (DTH) in X-irradiated Lewis rats, using pairs of analogs different in optical configuration of the COOH-terminal amino acid. The test cell line induced DTH against analogs with the COOH-terminal D-amino acid but not against those with the L-amino acid at the COOH terminus. A close correlation was found between the T cell line-induced DTH reaction in vivo and the proliferative response in vitro, in terms of clear discrimination of the optical configuration of COOH-terminal amino acid of disaccharide tetrapeptide. The L-isomers (non-stimulatory analogs of T cell proliferation) competitively inhibited the proliferation of the T cell line by the corresponding D-isomers. Thus the L-isomers appear to interact with Ia molecules on antigen-presenting cells. We conclude that COOH-terminal D-amino acid of the disaccharide tetrapeptide could be involved in binding to the T cell receptor, induction of T cell proliferation, and elicitation of DTH.

Effects of cytokines on cultured chondrocytes.

Progressive degradation of articular cartilage is a major feature of rheumatoid arthritis and osteoarthritis. Attention has focussed in recent years on some cytokines including interleukin 1 (IL-1) and tumor necrosis factor (TNF) . These cytokines caused cartilage to degrade its proteoglycan and inhibited the synthesis of new proteoglycan in explants of cartilage.We confirmed and extended these observations at the cellular level as follows. (1) Human recombinant IL-1α, IL-1β and TNFα strikingly inhibited the synthesis of glycosaminoglycan (GAG) and stimulated breakdown of proteoglycan in cultured chondrocytes. (2) These effects of IL-1α and TNFα were specifically neutralized by anti-IL-1α and anti-TNFα antisera, respectively. (3) Five different anti-IL-1α monoclonal antibodies, capable of neutralizing LAF activity to a varying degree, also neutralized IL-1α-induced inhibition of GAG synthesis and stimulation of proteoglycan breakdown. (4) The addition of TNFα, but not of IL-1 (α, β) to the cultures of chondrocytes stimulated DNA synthesis.In this review we related these results to the previous observations by others. Together these results suggest that IL-1 and TNFα may play an important role in the mediation of cartilage damage in chronic arthritis.

Partial characterization of thymocyte-activating factor derived from MDP-stimulated guinea pig fibroblasts.

The activity of fibroblast‐derived thymocyte activating factor (FTAF) of the guinea pig was measured, and the factor was partially characterized. The FTAF activity was heat labile, and destroyed by treatment with trypsin, chymotrypsin, and Streptomyces griseus protease, suggesting the protein nature of FTAF. FTAF bound to DEAE‐Sepharose CL‐6B in Tris‐HCl buffer at pH 8.0, and was eluted with 0.1–0.2 M NaCl. FTAF was absorbed with Blue Sepharose CL‐6B. The factor bound to a hydroxylapatite column in 10 mM phosphate buffer and was eluted in two major fractions, one fraction with 40 mM phosphate buffer, the other with 70–110 mM phosphate buffer. Finally, FTAF did not have as much effect on the proliferation of lymph node T cells as T‐cell‐activating monokines which exhibited marked stimulating effects on both T lymphocytes and thymocytes.

Use of the synthetic muramyl dipeptide for efficient induction of collagen arthritis and in vitro induction of a monokine, T cell activating factor

We developed an efficient method for production of experimental polyarthritis. Native collagen (type II) from human costal cartilage was emulsified with incomplete Freunds adjuvant (IFA) containing the synthetic adjuvant, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), and injected into one hind footpad of the PVG/c rat. This method reproducibly induced severe arthritis with high incidence, whereas the collagen alone in IFA produced mild arthritis with low incidence. MDP alone in IFA was shown to be minimally arthritogenic under identical conditions.MDP and its adjuvant-active analogs stimulated macrophages to produce a soluble factor (T cell activating factor, TFA) which was shown to be efficient in antigeninduced MIF production of immune lymphocytes as well as mitogen-induced proliferation of T lymphocytes. The in vitro system developed in this study should contribute to the understanding of the target cells for adjuvant activity of MDP. Moreover, the Mφ factor produced by stimulation with MDP may be advantageous for characterization of the factor, since MDP of low molecular weight appeared to be easily removed from the culture sup.