G. J. Thorbecke

Effect of thymus cell injections on germinal center formation in lymphoid tissues of nude (thymusless) mice

Abstract Nude mice, partially backcrossed to Balb/c or DBA/2, were injected iv with 5 × 10 7 thymus cells from the respective inbred strain. The response of these mice to immunization with Brucella abortus antigen was studied, with respect to both antibody production and the formation of germinal centers in their lymphoid tissues. The results were compared to those obtained with nude mice to which no thymus cells were given, as well as to Balb/c, DBA/2, or +/? litter mate controls. Nude mice formed less 19S as well as 7S antibody than did litter mate controls and completely lacked germinal centers in lymph nodes and gut-associated lymphoid tissue. Those nude mice which had been injected with thymus cells made a much better secondary response, both for 19S and for 7S antibody, and had active germinal centers in their lymph nodes as early as 3 wk after thymus cell injection. Intestinal lymphoid tissue in nude mice showed only slight reconstitution of germinal center activity several months after thymus cell injection and none at earlier times. Irradiated (3000 R) thymus cells appeared as effective as normal cells in facilitating germinal center appearance and 7S antibody production in the nude mice.

SOME HISTOLOGICAL AND FUNCTIONAL ASPECTS OF LYMPHOID TISSUE IN GERMFREE ANIMALS: I. MORPHOLOGICAL STUDIES*

Animals raised under germfree conditions can be considered to have much less contact with antigenic materials than animals raised in a normal environment. Since it is known that lymphoid tissue is concerned with the defense mechanism against infections, it can be expected that histological differences between germfree and control animals in this tissue will be found. In this paper I present the results of a comparative histological study of germfree and control animals. Particular attention was given to the occurrence of plasma cells and of secondary nodules in the lymphoid tissue. Department of Pathology, N e z York University College qf Medicine, hTew Yvrk, N . Y .

Amelioration of collagen-induced arthritis in DBA/1J mice by recombinant TSG-6, a tumor necrosis factor/interleukin-1–inducible protein

OBJECTIVE To examine the effect of recombinant TSG-6 on collagen-induced arthritis (CIA) in DBA/1J mice. TSG-6 is a tumor necrosis factor (TNF)/ interleukin-1 (IL-1)-inducible hyaluronan-binding protein produced by synovial cells and chondrocytes that is present in synovial fluids of patients with rheumatoid arthritis. METHODS To determine the effect of TSG-6 on chronic inflammatory joint disease, we induced CIA in DBA/1J mice by immunization with bovine type II collagen. Animals were treated with 12 intraperitoneal doses of 200 microg of recombinant TSG-6, beginning 3 days before the expected onset of disease symptoms. Progression of arthritis was monitored by determining the disease incidence, arthritis index, and footpad swelling. Levels of IgG1, IgG2a, and IgG2b antibodies against bovine and murine type II collagen and serum concentrations of IL-6 were determined at various time points. Histologic examination of affected joints was performed approximately 20 days after the onset of arthritis. RESULTS Treatment with recombinant TSG-6 protein had a potent ameliorative effect, manifested by decreases in the disease incidence, arthritis index, and footpad swelling. Histologic examination of affected joints in TSG-6-treated animals revealed little pannus formation and cartilage erosion, features which were conspicuous in control mice. Animals treated with recombinant TSG-6 developed significantly reduced levels of IgG1, IgG2a, and IgG2b antibodies against bovine and murine type II collagen. CONCLUSION The antiinflammatory effect of the TNF/IL-1-inducible TSG-6 protein in murine CIA suggests a role for this protein as an endogenous regulator of the inflammatory process.

Properties of reticulum cell sarcomas in sjl/j mice. Iii. Promotion of tumor growth in irradiated mice by normal lymphoid cells.

Abstract Previous irradiation (660–730 R) or cyclophosphamide treatment (400 mg/kg) of SJL/J mice markedly inhibited their ability to support growth of syngeneic intravenously (iv) injected reticulum cell sarcoma (RCS). Normal syngeneic lymphoid cells (5 × 10 7 ) injected iv after irradiation and 1 day prior to RCS cells partially restored the tumor growth in spleen and lymph nodes of irradiated mice as determined 5 to 7 days after RCS injection, but normal SJL/J serum injections had no effect. The cell type crucial for this effect appeared to be a macrophage, but some of the observations suggested the need for cell cooperation between T cells and a macrophage-like, bone marrow derived cell in this phenomenon. Pretreatment of tumor recipients with macrophage inhibiting agents, such as silica, trypan blue and carrageenan, also tended to inhibit tumor growth, particularly when combined with a dose of irradiation (460 R) which by itself had only a limited effect. However, macrophage stimulating agents did not protect against the irradiation effect.

Properties of reticulum cell sarcomas in SJL/J mice: IV. Minimal development of cytotoxic cells despite marked proliferation to syngeneic RCS in vivo and in vitro☆

Abstract Lymphoid cells from normal SJL/J mice gave high proliferative responses but failed to develop cytotoxic activity to γ-irradiated cells from syngeneic transplantable reticulum cell sarcomas (X-RCS). In spite of a vigorous in vivo proliferative response to X-RCS, cytotoxic activity was never generated to detectable levels in vivo. After repeated injections of X-RCS, spleen and, to a lesser degree, lymph node cells acquired the ability to give moderate secondary cytotoxic responses in vitro upon co-culture with X-RCS. This immunity was T-cell mediated and specific for RCS although it did not distinguish between different transplantable RCS lines. SJL/J mice also developed resistance to RCS growth after injection of X-RCS, which showed a transient RCS-line-specific component. (SJL/J × C57B1/6)F1 mice showed 60% less RCS growth than did SJL/J mice, and their lymphoid cells gave slightly lower proliferative responses than did cells from SJL/J mice, whereas (SJL/J × BALB/c)F1 mice showed little tumor growth, and their spleen cells proliferated only minimally to X-RCS. B10.S mice allowed moderate RCS growth. Cytotoxic activity was generated in co-cultures with X-RCS of immunized F1 spleen cells even after a single immunization in vivo but not in cultures of normal F1 cells with X-RCS.

Properties of reticulum cell sarcomas in sjl/j mice. II. Fate of labeled tumor cells in normal and irradiated syngeneic mice.

Abstract Transplantable reticulum cell sarcoma (RCS) cells were labeled with 3 H-uridine or 3 H-thymidine in vitro and injected intravenously into normal and irradiated syngeneic SJL/J mice. RCS cells exhibited typical B cell migration characteristics in peripheral lymphoid organs in both normal and irradiated recipients, localizing in follicles in a pattern resembling that of labeled normal bone marrow cells. However, over the first 72 hr after transfer, RCS cells diluted their label much less in irradiated than in normal recipients, reflecting their inability to proliferate in the irradiated hosts. The presence of unlabeled tumor cells did not significantly affect the distribution of labeled normal bone marrow or lymph node cells in the recipients. Thus, RCS fails to grow in irradiated recipients in spite of undisturbed homing characteristics and in the absence of any evidence of cytotoxic influences from the host.

Immunocompetent cells in the chicken: II. Synergism between thymus cells and either bursa or bone marrow cells in the humoral immune response to sheep erythrocytes

Newly hatched F1 hybrid chicks isogenic for the strong B histocompatibility locus were rendered immunologically incompetent by cyclophosphamide treatment and x-irradiation. They were then injected intravenously with thymus, bone marrow, or bursa cells together with sheep erythrocytes (SE) and received another iv injection of SE 3 days later. Splenic plaque-forming cells (PFC) and serum hemagglutinins were assayed 7 days after transfer. At donor ages of 14–26 days, cells from thymus (T) and bone marrow (BM) showed synergism when injected together, as indicated by a significantly higher geometric mean of PFC per recipient spleen in the BM + T group than in the BM group. The response of the T group was extremely low. With thymus and bursa cells from 6- to 28-day-old donors, significant synergism was demonstrated in 3 of 9 individual experiments. However, almost all the other 6 experiments showed marked differences in the same direction, and the combined probability for all experiments was < 0.001. The most striking demonstration of thymus + bursa synergism was made in 2 experiments using 1-week-old donors. Bone marrow cells from 1-week-old donors failed to cooperate with thymus, as did BM cells from older bursectomized agammaglobulinemic donors. This suggests that B cells from bone marrow originate in the bursa. Thymus-bursa cooperation was somewhat difficult to demonstrate in individual experiments using donors over 1 week of age, owing to the occurrence of some responses with bursal cells alone and to variability of response within bursa or bursa + thymus recipient groups. Synergism between thymus and bursa cells was more consistently demonstrable when irradiated normal spleen or low doses of bone marrow cells were added. These additions led to an increased response and a lowered coefficient of variation in the thymus + bursa recipient groups. The ‘third’ cell type needed for optimal response by the thymus and bursa cells together was tentatively identified as a macrophage.

Modulation by cytokines of induction of oral tolerance to type II collagen

OBJECTIVE To determine whether the simultaneous administration of drugs and/or cytokines such as transforming growth factor beta (TGFbeta) can render oral tolerance to type II collagen (CII) more effective in causing resistance to collagen-induced arthritis (CIA) in mice, and to investigate whether oral tolerance can still be induced when high levels of anti-CII are present. METHODS Tolerance was induced by intragastric feeding of low-dose CII to DBA/1 mice during a 2-week period, either before immunization with CII in Freunds complete adjuvant or after initiation of arthritis. Some mice were simultaneously injected with TGFbeta1 or with the H2 receptor agonist dimaprit. RESULTS Both TGFbeta1 and dimaprit increased the degree of oral tolerance obtained. TGFbeta1 augmented the induction of immunoregulatory CD8 T cells, which transferred the resistance to CIA induction to normal recipients. Feeding of CII for 2 weeks, starting after the onset of arthritis, still significantly ameliorated the course of CIA. CONCLUSION Administration of TGFbeta1 or dimaprit, both of which are believed to promote the development of immunoregulatory T cells, may reinforce induction of oral tolerance, even after the onset of arthritis.

When engineered to produce latent TGF-β1, antigen specific T cells down regulate Th1 cell-mediated autoimmune and Th2 cell-mediated allergic inflammatory processes☆

To determine whether T cells which produce large amounts of latent TGF-beta1 are capable of down-regulating autoimmune and allergic disease, myelin basic protein (MBP)-specific and ovalbumin (OVA)-specific BALB/c cloned Th1 cells were transduced with cDNA for murine TGF-beta1 by coculture with fibroblasts producing a genetically engineered retrovirus. The transduced MBP-specific Th1 cells were found to lose the capacity to provoke EAE in BALB/c mice, and to gain instead the ability to protect against EAE in (SJLxBALB/c) F1 mice immunized with proteolipid protein (PLP). This protective effect was not obtained with OVA-specific TGF-beta1 transduced Th1 cells. The transduced OVA-specific Th1 cells did protect against airway hyperreactivity induced by Th2-cell mediated responses to inhaled OVA. This effect was again antigen specific and it also could not be obtained with untransduced OVA-specific Th1 cells. In both cases these effects of antigen specific TGF-beta1 transduced T cells were nullified by administration of neutralizing anti-TGF-beta mAb. Thus, the antigen specificity of the cloned T cells allows the site-specific local delivery of therapeutic active TGF-beta1 to both Th1 and Th2 cell-mediated inflammatory infiltrates.

Induction of primary and inhibition of secondary antibody response to hapten by hapten conjugates of type III pneumococcal polysaccharide

Abstract Tri- or dinitrophenylated pneumococcal polysaccharide type III (TNP- or DNP SIII)) induced a primary 19S anti-TNP response without generating immunological memory to the hapten in LAF 1 mice. Hapten-hemocyanin (TNP-KLH) or hapten conjugates of B. abortus organisms (DNP-BA) induced both 19S and 7S primary responses and memory to the hapten. Spleen cells from mice immunized with TNP-KLH or DNP-BA did not give adoptive memory responses upon challenge with hapten-SIII and, in fact, were inhibited from responding to their homologous hapten conjugates by simultaneous injection of hapten-SIII. Incubation of TNP-KLH-primed spleen cells for as short as 5 min at 0 °C with 10 μg of TNP-SIII per milliliter virtually abolished their ability to give 19S and 7S memory responses to TNP-KLH upon transfer into irradiated recipients. It is suggested that a difference in avidity and/or number of anti-TNP receptors per cell between virgin and primed B cells may be an important factor in determining whether the cells will be stimulated or inhibited by exposure to hapten-SIII. Another factor may be a difference between virgin and memory cells in their requirement for T-cell help.