Collagen-induced arthritis in mice. VI. Synovial cells from collagen arthritic mice activate autologous lymphocytes in vitro

Synovial cells were extracted from normal and collagen-arthritic mice and investigated for lymphocyte-activating properties. In mixed cell culture, irradiated fibroblast-like synovial cells from DBA/1 LacJ arthritic mice stimulated a strong proliferative response in spleen cells from syngeneic normal mice, but not in cells from allogeneic DBA/2. B10.RIII, or BALB/c mice. This novel stimulus occurred in the absence of detectable Class II MHC antigen expression on the fibroblast-like synovial cell surface or increased autologous mixed lymphocyte reactions between DBA/1 LacJ spleen and lymph node cells. Irradiated synovial cells were also unable to present type II collagen to a collagen-specific T cell line and to stimulate proliferation. Addition of interferon-gamma or interleukin-1 failed to induce detectable surface Ia on the synovial fibroblasts or induce the capacity for antigen presentation in these cells.     

Adherent cell function in murine T lymphocyte antigen recognition. I. A. macrophage-dependent T cell proliferation assay in the mouse.

The data in this report describe a T cell proliferation assay with nylon wool column-purified murine lymph node lymphocyte from animals immunized by footpad injection of antigen in CFA. It was found that the in vitro immune response of sensitized T cells to soluble protein antigens was functionally dependent on the presence of adherent cells, more specifically macrophages, at all concentrations of in vitro antigen challenge. The response was due to T cells in that cytotoxic treatment of the immune lymphocyte cells with anti-Thy 1.2 serum and complement effectively eliminated the antigen-specific DNA synthetic responses. The antigen-specific proliferation of murine lymphocytes depleted of adhereent cells could not be reconstituted with either guinea pig macrophages nor murine fibroblasts, indicating the existence of species and cell type specificity. In contrast to previous observations in the guinea pig, soluble products of cultured adherent cells could at least partially replace the function of intact macrophages in the response to antigen.     

The B cell is the initiating antigen-presenting cell in peripheral lymph nodes

We have examined the role of B cells in antigen presentation in lymph nodes in several ways. We found that mice depleted of B lymphocytes via chronic injection of anti-mu-chain antibody do not mount peripheral lymph node T cell proliferative responses to normally immunogenic doses of antigen. Depletion of B cells by passage of immune lymph node cells over anti-immunoglobulin columns early after immunization depletes antigen-presenting function from draining lymph nodes, and this function can be restored by using B cells or splenic adherent cells to allow the remaining T cells to proliferate. Lymph node B cells present antigen very effectively to lines of antigen-specific T cells. However, unfractionated lymph node cells from anti-mu-treated mice present very poorly, if at all, whereas unfractionated spleen cells from the same mice do present antigen. This is in keeping with our previous finding that helper T cell function in the spleen is normal in B cell-deprived mice. Finally, when mice homozygous for the lymphoproliferative gene lpr are treated chronically with anti-mu-chain antibody, lymphadenopathy is greatly retarded, suggesting a role for B cells in the massive proliferation of T cells in this syndrome. From this analysis, it would appear that the initiating antigen-presenting cell in the lymph node is a B lymphocyte, and that B lymphocytes in lymph nodes may be distinct from those in the spleen. It is of interest that these results also suggest that the lymph node lacks an antigen-presenting cell that is found in the spleen, perhaps the dendritic cell.     

Regulation by adherent cells of macromolecular synthesis in lymphocyte populations.

The role of adherent cells in the regulation of lymphocyte DNA, RNA, and protein synthesis was investigated. Splenic adherent cells suppressed DNA synthesis of spleen cells. The reverse was true of lymph node adherent cells, i.e., DNA synthesis was less in the absence of adherent cells. Histocompatibility was not required for interaction between adherent and nonadherent cells. The regulatory adherent cell in either case was not θ positive. Removal of splenic adherent cells decreased RNA and protein synthesis of nonadherent spleen cells. RNA synthesis by lymph node cells increased when adherent cells were removed, but protein synthesis was lower. Autoradiographic analysis revealed that removal of adherent spleen cells allowed a greater number of nonadherent cells to respond to the presence of mitogen. Adherent cells were observed to regulate DNA synthesis of B lymphocytes also. Lymph node adherent cells amplified DNA synthesis in both nonadherent spleen and lymph node cells, while splenic adherent cells suppressed splenic nonadherent cells but stimulated nonadherent lymph node cells. We feel the data are compatible with the idea that at least two functionally distinct adherent cell populations exist. A mechanism is suggested to explain the data.     

Lymphocyte recognition of high endothelium: antibodies to distinct epitopes of an 85-95-kD glycoprotein antigen differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial endothelial cells

The tissue-specific homing of lymphocytes is directed by specialized high endothelial venules (HEV). At least three functionally independent lymphocyte/HEV recognition systems exist, controlling the extravasation of circulating lymphocytes into peripheral lymph nodes, mucosal lymphoid tissues (Peyer's patches or appendix), and the synovium of inflamed joints. We report here that antibodies capable of inhibiting human lymphocyte binding to one or more HEV types recognize a common 85-95-kD lymphocyte surface glycoprotein antigen, defined by the non-blocking monoclonal antibody, Hermes-1. We demonstrate that MEL-14, a monoclonal antibody against putative lymph node "homing receptors" in the mouse, functionally inhibits human lymphocyte binding to lymph node HEV but not to mucosal or synovial HEV, and cross-reacts with the 85-95-kD Hermes-1 antigen. Furthermore, we show that Hermes-3, a novel antibody produced by immunization with Hermes-1 antigen isolated from a mucosal HEV-specific cell line, selectively blocks lymphocyte binding to mucosal HEV. Such tissue specificity of inhibition suggests that MEL-14 and Hermes-3 block the function of specific lymphocyte recognition elements for lymph node and mucosal HEV, respectively. Recognition of synovial HEV also involves the 85-95-kD Hermes-1 antigen, in that a polyclonal antiserum produced against the isolated antigen blocks all three classes of lymphocyte-HEV interaction. From these studies, it is likely that the Hermes-1-defined 85-95-kD glycoprotein class either comprises a family of related but functionally independent receptors for HEV, or associates both physically and functionally with such receptors. The findings imply that related molecular mechanisms are involved in several functionally independent cell-cell recognition events that direct lymphocyte traffic.     

Failure of ovalbumin associated with allogeneic spleen ceels to stimulate proliferation of lymph node cells of immune mice.

Ovalbumin-pulsed spleen cells were found to stimulate thymidine uptake of lymph node cells of syngeneic mice immunized with ovalbumin in complete Freund's adjuvant after treatment of spleen cells with Mitomycin C but not after heating the spleen cells at 56degrees for 30 min. Ovalbumin-pulsed spleen cells of allogeneic mice failed to stimulate the immune lymph node cells more than unpulsed cells, although a net increase in the thymidine uptake above the allogeneic stimulation was observed when free ovalbumin was added to the mixed culture. To eliminate the high background of the mixed lymphocyte reaction, F1 mice were made chimeric with bone marrow of one of the parental strains. Using lymph node cells of the immunized chimeras, the stimulation by pulsed spleen cells was much greater when antigen was presented on cells of the parental strain used for bone marrow injection than when presented on cells of the other parental strain.     

Cytotoxic action of immune lymphocytes on "adherent" cells (macrophages) of autologous system lymph nodes during delayed hypersensitivity]

A study was made in the autologous system of the cytotoxic action of immune lymphocytes on the "adherent" cells (macrophages) of the lymph nodes of guinea pigs in delayed hypersensitivity (DH) to the streptococcus antigens and tuberculoproteins. Death of a considerable number of the "adherent" cells in cultivation of a suspension of cells of the lymph nodes of animals sensitized with the culture of streptococcus or BCG in the presence of specific antigen (thermo-stable streptococcus fraction or tuberculin, respectively) alone. Detection of death of the "adherent" cells in the autologous system can be used as one of specific and sensitive tests in studying the DH.     

Tumour-associated proliferative responses in vitro of regional lymph nodes draining solid cancers in man

Summary The proliferative responses in vitro of tumourdraining lymph node lymphocytes were evaluated against autologous colon and lung carcinoma cells. The reactivity of lymphocytes appeared to be directed against tumour-associated rather than tumour-specific antigens. The lymphocyte reactivity detected was not due to an autologous mixed lymphocyte reaction. Recombinant interleukin-2 augmented the responses detected but not their tumour specificity. Phenotypic characterisation indicated the presence of T suppressor/cytotoxic (TS/C) cells as well as natural killer (NK) cells. Only the latter, however, were active in functional cytotoxicity assays. The inability to generate both tumour-specific proliferation of tumour-draining lymph node lymphocytes and tumour-specific cytotoxic killer cells may be due to the presence of suppressor cells in the regional lymph nodes; preliminary data suggest the presence of such cells.Part of this project was supported by a grant from Cancer Research Campaign     

Granulocyte-macrophage colony-stimulating factor augments the primary antibody response by enhancing the function of antigen-presenting cells

Purified, recombinant-derived murine granulocyte-monocyte colony-stimulating factor was found to enhance the primary in vitro immune response to SRBC by murine spleen cells. In determining the mechanism of this augmentation, it was found that only splenic adherent cells and neither resting nor activated T cells nor B cells expressed specific receptors for GM-CSF. When splenic adherent cells were pulsed briefly with GM-CSF before addition to macrophage-depleted cultures, they reconstituted the PFC response to a significantly greater degree than did control macrophages. Splenic adherent cells incubated overnight with SRBC plus GM-CSF were also more efficient antigen-presenting cells than splenic adherent cells incubated with antigen alone. The mechanism of this enhanced antigen presentation was found to be due to a GM-CSF-dependent increase in the level of IL 1 secretion and Ia antigen expression. Consistent with these data was the finding that GM-CSF augmented IL 2 production by splenic T cells in response to suboptimal concentrations of Con A. Finally, the day 5 in vivo antibody response (as measured by serum titers) of mice immunized with a low dose of SRBC was enhanced by two daily inoculations of GM-CSF. Thus, the role that GM-CSF plays in augmenting immune responses may not be solely accounted for by its ability to cause the proliferation or differentiation of macrophages, but more than likely includes its ability to enhance the function of antigen-presenting macrophages.     

Age-related changes, including synergy and suppression, in the mixed lymphocyte reaction in long-lived mice.

The mixed lymphocyte culture reaction represents the in vitro counterpart of the recognition phase of the graft-versus-host reaction, and of allograft rejection. The mixed lymphocyte culture reactivities of lymph node and spleen cells from all strains show a striking decline with advanced age. Furthermore, studies of "synergy" between subpopulations of T cells in the mixed lymphocyte culture reaction suggest that the cells of the recirculating lymphoid pool (T2 cells) in particular display a functional decline. Finally, spleen cells from old mice of appropriate strains inhibit or suppress the mixed lymphocyte culture reactivity of lymph node or spleen cells from young mice.     

Trypanosoma cruzi: Responses by cells from infected mice to alloantigens

In unidirectional mixed lymphocyte cultures containing (as responders, stimulators, or regulators) spleen cells from mice infected with Trypanosoma cruzi, alloantigen responses were less than in cultures containing normal spleen cells only. Depletion of plastic adherent cells from infected spleen cells (stimulators or regulators) reversed their inhibitory effect on normal spleen cells (responders); removal of adherent responder cells and/or B lymphocytes did not alter the low alloantigen responses of normal spleen cells (stimulated by infected spleen cells) or infected spleen cells (stimulated by normal spleen cells). Infected spleen cells were effective in regulating mixed lymphocyte cultures only when added at the initiation of the culture. Serum from infected mice suppressed mixed lymphocyte cultures containing responder spleen cells syngeneic to the serum donor if added up to 24 hr after initiation of cultures, whereas the “suppressor serum” had to be present at the initiation of cultures when responder cells were allogeneic to the serum donor. Cultures of infected spleen cells (whole or macrophage enriched) produced a factor which was suppressive when added to mixed lymphocyte cultures containing syngeneic responder cells at initiation. It is proposed that the serum suppressor substance regulates cell-mediated immune responses directly by suppressing the response-potential of cells and indirectly by triggering the release of a factor from adherent splenic cells which induces a hyporesponsive state in T lymphocytes.     

A subpopulation of adherent accessory cells bearing both I-A and I-E or C subregion antigens is required for antigen-specific murine T lymphocyte proliferation.

A murine T lymphocyte proliferation assay that used antigen-primed lymph node T cells, was antigen specific, and required exogenous accessory cells was used to characterize the accessory cells that supported proliferation. These cells were Thy 1.2 negative, radioresistant, glass-adherent, and were functional only if alive. The accessory cell function of spleen adherent cells was much greater than that of peritoneal cells. Also, the accessory cell function of spleen adherent cells was proportional to the length of time such cells were incubated with antigen and very small numbers of such cells provided accessory cell function. Cytotoxic studies with subregion-restricted anti-Ia antibodies and complement indicated that accessory cell function resided in a subpopulation of spleen adherent cells that bore both I-A and I-E or C subregion antigens. The function of such cells was not related to a selective ability (vs other spleen adherent cells) to take up antigen. These data indicate that antigen-specific stimulation of T lymphocyte proliferation requires at least one specific subpopulation of spleen adherent cells that can be phenotypically identified by its expression of Ia antigens and are consistent with the possibility that Ia antigens may be Ir gene products.     

Immunophenotypic and cell cycle analysis of lymph node cells from dimethylbenzanthracene-treated mice

The chemical carcinogen 7, 12-dimethylbenz-(a)anthracene (DMBA) depletes Langerhans cells from murine epidermis. Application of contact sensitizers to DMBA-treated skin induces specific immunological tolerance due to a DMBA-resistant epidermal antigen presenting cell (APC) migrating to local lymph nodes where it presents antigen in a way which activates suppressor cells. As alterations in local lymph node lymphocytes may enhance the ability of the DMBA-resistant APC to activate suppressor cells, these cells were examined in DMBA-treated mice. Lymph nodes in DMBA-treated mice had normal morphology but were larger and contained increased numbers of lymphocytes. Cell cycle analysis revealed that these lymphocytes did not arise from division within the lymph node, suggesting alterations in homing properties of lymphocytes. Contact sensitizer applied to DMBA-treated skin did not increase lymphocyte division, possibly due to suppressor cell inhibition of the development of effector lymphocytes. DMBA treatment had no effect on B cells or Ia expression, but decreased levels of the T lymphocyte cell surface molecule Thy-1, and increased L3T4 and Lyt-2 as quantitated by flow cytofluorimetry. These changes could influence the development of immune responses as these T cell molecules are receptors involved in lymphocyte interactions.     

Immunophenotypic and cell cycle analysis of lymph node cells from dimethylbenzanthracene-treated mice

The chemical carcinogen 7, 12-dimethylbenz(a)anthracene (DMBA) depletes Langerhans cells from murine epidermis. Application of contact sensitizers to DMBA-treated skin induces specific immunological tolerance due to a DMBA-resistant epidermal antigen presenting cell (APC) migrating to local lymph nodes where it presents antigen in a way which activates suppressor cells. As alterations in local lymph node lymphocytes may enhance the ability of the DMBA-resistant APC to activate suppressor cells, these cells were examined in DMBA-treated mice. Lymph nodes in DMBA-treated mice had normal morphology but were larger and contained increased numbers of lymphocytes. Cell cycle analysis revealed that these lymphocytes did not arise from division within the lymph node, suggesting alterations in homing properties of lymphocytes. Contact sensitizer applied to DMBA-treated skin did not increase lymphocyte division, possibly due to suppressor cell inhibition of the development of effector lymphocytes. DMBA treatment had no effect on B cells or Ia expression, but decreased levels of the T lymphocyte cell surface molecule Thy-1, and increased L3T4 and Lyt-2 as quantitated by flow cytofluorimetry. These changes could influence the development of immune responses as these T cell molecules are receptors involved in lymphocyte interactions.     

Macrophage activation of allogeneic lymphocyte proliferation in the guinea pig mixed leukocyte culture.

The role of the macrophage in the guinea pig mixed leukocyte culture was investigated. Macrophages obtained from oil-induced peritoneal exudates, peritoneal wash-out cells, spleen, and alveolar washings were found to be effective stimulators of allogeneic lymph node and splenic lymphocyte DNA synthesis. The stimulatory properties of macrophages proved radioresistant but viability dependent. Unfractionated lymph node cells or adherence column purified lymph node lymphocytes and thymocytes were only minimally active as stimulators, even in the presence of macrophages syngeneic to the responder lymphocytes. Allogeneic fibroblasts, polymorphonuclear leukocytes, L2C leukemia cells, and xenogeneic (murine) macrophages failed to simulate. These data provide evidence that the macrophage is the predominant stimulator of the mixed leukocyte culture in the guinea pig.     

The migration of lymphocytes into rat popliteal lymph nodes during antigenic promotion or competition between heterologous erythrocytes

The injection of chicken and sheep red blood cells (CRBC and SRBC) into rat popliteal lymph nodes either together or sequentially 2, 4, 6, or 8 days apart resulted in an enhanced immune response when the second antigen was injected 2 or 4 days after the injection of the first antigen (antigenic promotion) or a suppressed immune response when the second antigen was injected 6 days after the injection of the first antigen (antigenic competition). The immune response to either antigen was dependent upon the time of administration of the second antigen with respect to the first antigen. Lymphocyte migration into antigenically stimulated lymph nodes was greater when the two antigens were injected sequentially rather than together. Further, the migration of lymphocytes into the lymph node was enhanced when the second antigen was injected during the inductive or suppressive phase of the immune response to the first antigen (CRBC) regardless of whether the same (CRBC) or an antigenically unrelated antigen (SRBC) was used as the second antigen. While antigenic promotion may in part be explained by the increased rate at which lymphocytes migrate into lymph nodes, lymphocyte migration is also enhanced during antigenic competition. This suggests that while suppressor cells/factors may regulate the effector phase of an immune response they do not directly modulate the migration of blood-borne lymphocytes into the lymph node.     

T cell proliferation induced by autologous non-T cells is a response to apoptotic cells processed by dendritic cells

Self-reactive T cells are present in the mature immune repertoire as demonstrated by T cell proliferation induced by autologous non-T cells in the autologous mixed lymphocyte reaction. This reaction generates regulatory T cells in vitro and may reflect immune regulatory pathways in vivo, but the antigenic peptides recognized remain uncharacterized. We revisited this issue in light of the importance of apoptosis in immune regulation. We found that apoptosis among peripheral blood non-T stimulator cells is associated with augmented induction of autologous T cell proliferation. Our data show that caspase activity in the non-T stimulator population is essential for induction of autologous T cell proliferation, suggesting that cellular components in the non-T cell fraction are enzymatically modified, most likely by effector caspases, and have a direct or indirect effect on autoreactive T cell activation. Furthermore, exposure of macrophage-derived dendritic cells to apoptotic non-T cells augments autologous T cell proliferation, and blockade of alpha(v)beta(5) integrin, but not alpha(v)beta(3), inhibits the capacity of irradiated non-T cells or dendritic cells to stimulate autologous T cell proliferation. These experiments, using an entirely autologous system, suggest the interpretation that autoreactive T cells may recognize self-Ags modified through the actions of caspases and presented to T cells by dendritic cells. Induction of an in vivo autologous mixed lymphocyte reaction by caspase-modified self-Ags present in apoptotic cells may represent a mechanism to maintain peripheral immune tolerance.     

Characterization of rat T cell subset antigen by monoclonal antibody

6B2-B8 T cell hybridoma cells were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated RTH-7, reacted with 89.5% of rat thymocytes, 30.2% of rat spleen cells, and 42.3% of rat lymph node cells. The RTH-7 reacted with a subset of rat T cells but not with B cells. Double staining analysis demonstrated that RTH-7 stained a rat T cell subset distinct from R1-10B5-positive cells that were known to be equivalent to mouse Lyt-2. It was revealed that RTH-7 and W3/25 recognize different antigenic epitopes on the same molecule. The RTH-7 as well as W3/25 substantially inhibited the production of interleukin 2 by cells in mixed lymphocyte reaction and the lymphocyte proliferation induced by mixed lymphocyte reaction. The RTH-7 inhibited the lymphocyte proliferation induced by Con A whereas W3/25 failed to do so. The RTH-7 defined antigen has a molecular weight of 53,000 under reducing condition and 47,000 under nonreducing condition. The RTH-7 defined antigen showed a wide range of heterogeneity in pI (6.2-8.8). The associated molecule of approximate molecular weight of 27,000 was occasionally detected with the RTH-7 defined antigen in 6B2-B8 T cell hybridoma cells as well as peripheral T cells but not in thymocytes. Thus, RTH-7 detects a cell surface antigen of a functional T cell subset of rat origin.     

Regional influences on the physical properties of T cell membranes

Differences in the composition of membrane lipids are well documented between cells from distinct tissues. These differences may be manifested by changes in the motional freedom or fluidity of lipid molecules within plasma membranes and may predispose to alterations in cellular function. Regional influences on immune function have been implied by the finding that thymic-derived cells from murine spleen and lymph nodes are differentially responsive to antigen priming. The possibility that microenvironment also shapes the physical properties of T lymphocyte membranes has not been explored and is the focus of this study. Using mice as the experimental model, differences were found in fluidity and in the resting level of intracellular free-ionized Ca2+ between splenic and lymph node T cells from immunologically normal mice and from autoimmune-prone MRL-lpr/lpr mice. The results indicate that T cells are more heterogeneous than previously recognized and suggest a potential role for microenvironment in determining immune responsiveness.     

Lymphocyte transformation induced by autologous cells.

Human peripheral blood T lymphocytes are stimulated to proliferate when cultured with autologous B-lymphoblastoid cell lines, autologous mitogen-induced lymphoblasts, or autologous non-T blood lymphocytes. This reaction, the autologous mixed lymphocyte reaction, has attributes of an immune response possessing both memory and specificity. The capacity to stimulate autologous T lymphocyte proliferation depends on the lineage of the lymphoid cell and not on its establishment in continuous culture or carriage of the EB viral genome. The determinant on non-T lymphocytes which stimulates the autologous mixed lymphocyte reaction appears to be an Ia determinant. Thus, allogeneic graft rejection and the allogenic mixed lymphocyte reaction are very likely extensions of an immune response expressed within the host.