Lymph Node Cell Cultures Research Articles

Uveitogenicity Is Associated with a Th1-like Lymphokine Profile: Cytokine-Dependent Modulation of Early and Committed Effector T Cells in Experimental Autoimmune Uveitis

This study addresses the nature of the pathogenic effector T cell in experimental autoimmune uveoretinitis and the effect of different cytokines on these cellsin vitro.Lymph node cells of B10.RIII mice immunized with the uveitogenic peptide 161-180 of interphotoreceptor retinoid binding protein were cultured with the peptide with or without IL-12, IL-4, or anti-IL-4. An antigen-specific T cell line was subsequently derived from these cells. Primary cultures of immune lymph node cells stimulated with the peptide proliferated and produced IL-2 and some IL-4, but no IFN-γ. The addition of recombinant IL-12 resulted in abundant production of IFN-γ, which was blocked by the addition of IL-4 and was enhanced by anti-IL-4. Only those cultures that produced IFN-γin vitrowere uveitogenicin vivo.A long-term uveitogenic T cell line, initially derived in the presence of IL-12, produced IFN-γ and IL-2, but not IL-4, and was CD4+(Th1-like). Antigen-specific proliferation and IFN-γ production of the line were enhanced by exogenous IL-4, TGF-β, IL-2, IL-6, IL-7, and IL-9 and were inhibited by IL-10 and TNF-α. Our results provide support for the hypothesis that the uveitogenic effector T cell has a Th1-like phenotype. Furthermore, the data suggest that the effects of the cytokine milieu on fully differentiated Th1 effectors may differ considerably from their effects on less mature stages of antigen-specific T cells.

Antiinterleukin-5 antibody prevents airway hyperresponsiveness in a murine model of airway sensitization.

Eosinophils play a central role in the inflammatory response associated with bronchial asthma. We studied the involvement of eosinophils in the development of airway hyperresponsiveness (AHR) in a mouse model of allergic airway sensitization. Sensitization of BALB/c mice to OVA via the airways induced allergen-specific T-cell responses, IgE production, immediate cutaneous hypersensitivity (ICH), and increased airway reactivity. Airway sensitization was associated with eosinophil infiltration of the airways and increased production of interleukin-5 (IL-5) in cultures of peribronchial lymph node cells. Treatment of OVA-challenged animals with anti-IL-5 antibody during the sensitization protocol completely abolished the infiltration of eosinophils into the lung tissue and prevented the development of AHR without affecting levels of allergen-specific IgE, cutaneous hypersensitivity and allergen-specific T cell responses. These findings demonstrate that infiltration of lung tissue by eosinophils, triggered by increased IL-5 production, is a major factor in the development of AHR in this mouse model of airway sensitization.

Fine mapping of T-cell determinants of bovine beta-lactoglobulin.

T-cell recognition sites, i.e. T-cell determinants, of bovine beta-lactoglobulin, a major allergen in milk, were analyzed in detail. For this purpose, we prepared primary cultures of lymph node cells from three strains of mice, C57BL/6 (H-2b), C3H/HeN (H-2k), and BALB/c (H-2d), and examined the proliferative response of these cells to a complete set of overlapping 15-mer peptides which covered the entire sequence of beta-lactoglobulin by shifting in single amino acid steps. We were able to determine the putative core sequence of each T-cell determinant and estimate its relative importance. In the case of C57BL/6 mice, dominant, subdominant, and minor determinants were identified as residues 122-130, 16-26, and 108-122, respectively, as represented by their core sequences. Each determinant peptide induced the production of interferon-gamma, the amount of which showed a correlation with the intensity of the proliferative response induced by each determinant. In the case of C3H/HeN mice, a dominant determinant comprised of residues 140-148 was identified together with three subdominant and two minor determinants. Dominant T-cell determinants recognized in BALB/c mice were identified as residues 67-75, 71-79, and 80-88, and six other regions were identified as subdominant determinants. Comparisons between our results and the determinants predicted from relevant MHC-binding motifs reported to date revealed the inadequacy of the motifs in predicting even the dominant determinants. The information obtained by complete mapping of T-cell determinants as done in this study is expected to be helpful in establishment and evaluation of new prediction methods and also may contribute to the development of a new approach to control immune responses by manipulation of the T-cell determinants of allergens.

Immunoregulatory effects of glandular kallikrein from the salivary submandibular gland of rats.

A protein of 40 kD molecular weight was isolated from the salivary submandibular glands of male rats. The protein catalyzed the hydrolysis of alpha-N-benzoyl-L-arginine ethyl ester. This esterase activity was inhibitable with the protease inhibitor aprotinin. The sequence of the first 25 amino acids of this protein was identical to that of rat glandular kallikrein (rGK). When added to cultures of murine lymph node cells suboptimally stimulated with the T cell mitogen concanavalin A, rGK markedly stimulated the proliferative activity of these cells. When injected into mice, rGK suppressed the contact sensitivity response to picryl chloride, a form of delayed-type hypersensitivity. Similar in vitro and in vivo effects were induced with GK from porcine pancreas (pGK). Moreover, the aforementioned in vitro and in vivo effects were abolished by aprotinin either added to the tissue culture medium or injected into the animals immediately before rGK or pGK. This demonstrates that the enzymatic activity of rGK and pGK is important for the induction of immunoregulatory effects. These results suggest that rGK is a systemic immunoregulatory enzyme with immunosuppressive potential. GK is the first example for systemic immunoregulation by an enzyme, the secretion of which is under neuroendocrine control.

Failure of aged rats to accumulate eosinophils in allergic inflammation of the airway

To investigate the effect of aging on the allergic airway response, we examined the bronchoconstrictive responses and cellular inflammatory changes in a rat model of bronchial asthma by evaluating young and old animals. Two different age groups of Brown-Norway rats, actively sensitized by injection of ovalbumin into the foot pads, were used: 7 to 8 weeks old (young group) and 100 to 120 weeks old (aged group). Both the aged and young rats produced an ovalbumin-specific IgE antibody and exhibited an immediate asthmatic response after exposure to ovalbumin, but the degree of specific IgE antibody was significantly higher in young rats. The young group showed a marked increase in the number of eosinophils and neutrophils in bronchoalveolar lavage fluid 2 days after exposure to ovalbumin, whereas no eosinophilia was seen in the aged group. To evaluate the mechanism of the decreased accumulation of eosinophils in aged rats, cells from popliteal lymph nodes from ovalbumin-sensitized rats were incubated with ovalbumin for 48 hours. Although eosinophil chemotactic activity, determined by a modified Boyden chamber method, was present in the supernatant of cultured lymph node cells from young rats, it was absent from those of aged rats. In vivo administration of anti-IL-5 monoclonal antibody revealed that one of the factors of eosinophil chemotactic activity was IL-5. Lymph node cells from aged rats tended to produce greater amounts of interferon-γ than did those from young animals. Findings indicate that aged rats have a defect in eosinophil accumulation in sites exposed to antigen, probably because of an age-dependent alteration in T cells. (J Allergy Clin Immunol 1997;99:38-47.)

Saponin adjuvant primes for a dominant interleukin-10 production to ovalbumin and to Trypanosoma cruzi antigen.

The adjuvant activity of saponin for T-cell responses was evaluated and compared with that of complete Freund's adjuvant (CFA) in two antigen systems: a lysate of the protozoa Trypanosoma cruzi and ovalbumin (OA). Strong delayed-type hypersensitivity and T-cell proliferate responses, comparable with those stimulated by CFA, were observed for both antigens following immunization with saponin as adjuvant. Upon in vitro secondary antigen stimulation, high interleukin-10 (IL-10) and low interferon-gamma (IFN-gamma) levels were observed in lymph node (LN) cell cultures from saponin-immunized mice in contrast with the high IFN-gamma and decreased IL-10 production by LN cells from CFA-immunized mice. Production of IL-10 and IFN-gamma in these conditions was CD4-activation dependent. Concanavalin A (Con A)-stimulated interleukin-4 (IL-4) production was higher in saponin-immunized mice than in CFA-immunized mice. IL-10 produced by LN cells from saponin-immunized mice suppressed IFN-gamma production and Con A-induced proliferation. Taken together, these data are consistent with in vivo stimulation of both T-helper (Th)1 and Th2-type cells by immunization with saponin; in vitro a Th2-type cytokine response with high IL-10 production predominates, indicating preferential priming towards a Th2-type response. Immunization with CFA induced a Th1-type cytokine response. To our knowledge this is the first report in which an adjuvant is shown to prime for a dominant IL-10 production.

Extrathymic Development of Self-Reactive γδ T Cells in Athymic BALB/cnu/nuMice

In the present report, we demonstrated that extrathymic γδ T cells proliferated in the mixed culture of lymph node cells of BALB/cnu/numice and irradiated syngeneic spleen cells (syngeneic MLR). Reverse transcription–polymerase chain reaction (RT-PCR) analyses revealed that Vδ5 and Vδ6 were dominantly expressed by the γδ T cells from the syngeneic MLR. Furthermore, the syngeneic MLR may expand Vδ5+T cells with BALB invariant delta (BID) sequence which is specifically detected in BALB background mice because Vδ5 RT-PCR products from syngeneic MLR were digested with anAluI restriction enzyme which cut the Dδ2–Jδ1 junction of the BID sequence but not those of most non-BID Vδ5 sequences. Interestingly, anti-mycobacterial and mammalian heat shock protein (Hsp) 60 mAb ML30 suppressed the generation of Vδ5 with anAluI site in syngeneic MLR. These results suggest that extrathymically derived Vδ5+and Vδ6+γδ T cells recognize BALB/c self antigens and that the Hsp60 or an Hsp60-related molecule may be involved in the anti-self response of Vδ5+BID+γδ T cells.

Relationship between Th1/Th2 cytokine patterns and the arthritogenic response in collagen-induced arthritis.

It is hypothesized that the balance of cytokines produced by Th1/Th2 subsets of T helper cells plays an important role in the development of autoimmune diseases. Murine collagen-induced arthritis (CIA) is an example of an autoimmune disease in which immunization with cartilage-derived type II collagen induces, firstly, a T cell response to type II collagen and, secondly, the manifestation of a destructive inflammatory response in affected joints. We have investigated the role of Th1/Th2 responses in the development of CIA by monitoring levels of interferon (IFN)-gamma (a Th1 cytokine) and interleukin (IL)-4 and IL-10 (Th2 cytokines), and IL-1 beta and tumor necrosis factor (TNF) (pro-inflammatory cytokines) produced by cultured draining lymph node cells (LNC) from collagen-immunized DBA/1 mice during the induction phase of arthritis and throughout the time of clinical manifestation and subsequent remission of the disease. Although a transient increase in IL-10 was detected 3 days after immunization, Th2 cytokine production was found to be almost completely suppressed 6 days after immunization. In contrast, IFN-gamma was detected in LNC cultures as early as 6 days after immunization and the addition of type II collagen to the culture medium resulted in an approximately 10-fold increase in IFN-gamma production, indicating that a predominantly Th1 response had become established by this time. IFN-gamma production by LNC was found to be further increased at the time of clinical manifestation of arthritis and could be up-regulated by co-culture with type II collagen. IL-10 was not detected in LNC cultures at the onset of arthritis and IL-4, although present, was found to be markedly suppressed in LNC cultures containing type II collagen. These findings indicate that Th1 responses are predominant at the time of onset of arthritis and that the activation of collagen-specific Th1 cells may result in suppression of Th2 activity. IFN-gamma production declined progressively during the progression and subsequent remission of arthritis whereas levels of IL-10 increased and low, though persistent, levels of IL-4 were detected throughout this period. High levels of IL-1 beta and TNF-alpha production were detected at the onset of the disease. The role of Th1 responses in the development of CIA was further emphasized by the observation that immunization of mice with type II collagen in incomplete Freund's adjuvant, which normally fails to induce arthritis, resulted in a predominantly Th2 cytokine profile.

Specific immunotherapy with tumour-draining lymph node cells cultured with both anti-CD3 and anti-CD28 monoclonal antibodies.

For providing costimulatory signals, we utilized anti-CD28 monoclonal antibody (mAb) for the in vitro culture of tumour-draining lymph node (LN) cells. The proliferation of B16 melanoma-draining LN cells in the culture with anti-CD3 mAb was remarkably enhanced by the addition of anti-CD28 mAb. In culture with both anti-CD3 and anti-CD28 mAb, the B16-draining LN cells produced a higher level of interferon-gamma, but not interleukin-4, than with anti-CD3 mAb alone. The B16-draining LN cells efficiently expanded in the culture with both anti-CD3 and anti-CD28 mAb and subsequently with a low dose of IL-2 (anti-CD3 plus anti-CD28/IL-2). The expanded cells consisted predominantly of CD8+ T cells and showed a specific cytolytic activity, in a major histocompatibility complex (MHC) class I-restricted manner, even without in vitro restimulation. In addition, the adoptive transfer of the B16-draining LN cells, expanded in the culture protocol of anti-CD3 plus anti-CD28/IL-2, showed a significant anti-tumour effect against metastatic B16 melanoma in combination with IL-2. The cured mice thus acquired a specific protective immunity. Moreover, this protocol was also moderately effective against poorly immunogenic 3LL carcinoma. Overall, our results suggest the potential for another immunotherapeutic strategy based on 'the costimulatory theory' other than vaccination with B7-transfected tumour cells.

T cell populations primed by hapten sensitization in contact sensitivity are distinguished by polarized patterns of cytokine production: interferon gamma-producing (Tc1) effector CD8+ T cells and interleukin (Il) 4/Il-10-producing (Th2) negative regulatory CD4+ T cells.

Contact hypersensitivity (CHS) is a T cell-mediated response to hapten sensitization of the epidermis. The roles of CD4+ and CD8+ T cells in CHS have remained unclear, however, as studies to define either subset as the T cells mediating CHS have provided conflicting results. The goal of this study was to correlate the in vivo function of CD4+ and CD8+ T cells in CHS with the cytokines produced by each T cell population. Antibody-mediated depletion of CD4+ T cells before sensitization of BALB/c mice with 2,4-dinitrofluorobenzene (DNFB) or oxazolone (Ox) resulted in increased and prolonged CHS responses, indicating CD4+ T cells as negative regulators of the response. Depletion of CD8+ T cells resulted in low or abrogated responses, indicating CD8+ T cells as the effector cells in CHS. Sensitization with DNFB or Ox induced lymph node cell populations of CD8+ T cells producing interferon (IFN)-gamma and no interleukin (Il) 4 or Il-10, and CD4+ T cells producing Il-4 and Il-10 and no or little detectable IFN-gamma. The polarized patterns of cytokine production were stimulated by culture of hapten-primed lymph node cells either on anti-T cell receptor antibody-coated wells or with semipurified Langerhans cells isolated from hapten-sensitized mice. Stimulation of cytokine production during culture of hapten-primed CD4+ or CD8+ T cells with Langerhans cells was hapten specific and restricted to class II or class I major histocompatibility complex, respectively. The induction of the CD4+ and CD8+ T cells producing the polarized patterns of cytokines was not restricted to BALB/c mice, as cells from Ox sensitized C57B1/6 and B10.D2 mice produced the same patterns. Collectively, these results expose the induction of two polarized and functionally opposing populations of T cells by hapten sensitization to induce CHS: IFN-gamma-producing effector CD8+ T cells and Il-4/Il-10-producing CD4+ T cells that negatively regulate the response.

IL-12, as an adjuvant, promotes a T helper 1 cell, but does not suppress a T helper 2 cell recall response.

IL-12 is a potent inducer of NK and cytolytic T cell activity, IFN-gamma production, and T cell proliferation, and is necessary for differentiation of naive T cells to the Th1 subset. We have previously shown that IL-12 promotes a primary Th1 response and suppresses a primary Th2 response in lymph nodes of mice primed with a model hapten-protein conjugate, 2,4,6-trinitrophenyl (TNP)-keyhole limpet hemocyanin (KLH). We have now extended these studies to determine the Th phenotype of the recall response following immunization with soluble Ag and IL-12. For these experiments, mice were primed with TNP-KLH with or without treatment with IL-12, allowed to progress beyond the primary immune response, and challenged by i.p. injection of TNP-KLH. The phenotype of the recall response was monitored by measuring ex vivo production of IFN-gamma and IL-4 in Ag-stimulated lymph node and spleen cell cultures. Titer and isotype of TNP-specific serum Abs were also evaluated. Mice primed with Ag+IL-12 developed a Th1 recall response, as detected by KLH-specific IFN-gamma production from cultured spleen cells and the presence of TNP-specific IgG2a Ab in serum. However, they also developed an Ag-specific Th2 recall response, as characterized by Ag-induced IL-4 production from spleen cells and the presence of high titers of anti-TNP IgG1 in the serum. Studies of the cytokine profile during the primary response revealed that IL-12 induced in spleen cells the capacity to express both IL-4 and IFN-gamma. CD4+ T cells are necessary for production of IL-4 in the spleens of IL-12-treated mice, and most likely account for the Th2 recall response detected in mice primed with Ag+IL-12. These results indicate that the Th1 phenotype induced by immunization with IL-12 and Ag is maintained so that a Th1 recall response is expressed upon subsequent challenge with Ag. However, immunization with IL-12 also supports the development of a Th2 recall response, indicating that the Th1-inducing effect of IL-12 in vivo is not accompanied by a long lasting suppression of Th2 development.

Trypanosoma congolense: Proliferative Responses and Interleukin Production in Lymph Node Cells of Infected Cattle

T-cell-mediated immune responses to defined antigens of Trypanosoma congolense were measured in cattle undergoing primary infection. The antigens used were the variable surface glycoprotein and two invariant antigens, a 33-kDa cysteine protease (congopain) and a recombinant form of a 69-kDa heat-shock protein. Proliferative responses were highest during the second week postinfection and were detected in cells obtained from the lymph node draining the site of infection but not in peripheral blood mononuclear cells. Production of IL-2 and IFN-γ was measured in supernatants from antigen-stimulated lymph node cell cultures. Expression of IL-2, IL-4, and IFN-γ mRNA was detected in antigen-stimulated lymph node cells by reverse transcription-polymerase chain amplification.

Endogenous IL-12 is required for control of Th2 cytokine responses capable of exacerbating leishmaniasis in normally resistant mice.

We investigated the mechanisms by which treatment with anti-IL-12 Ab prevents cure of infection with Leishmania major in resistant C57BL/6 mice. Consistent with delayed production of IL-12, anti-IL-12 Abs could be administered as late as 2 wk after infection to exacerbate disease. Starting at 2 wk of infection, the cultured lymph node cells from mice treated with either polyclonal or monoclonal anti-IL-12 Abs persistently generated 3- to 10-fold more IL-4 and IL-10 in response to L. major Ag compared with cells from mice receiving preimmune goat IgG. Reciprocal decreases in Ag-specific IFN-gamma production were observed in mice receiving anti-IL-12 Abs. A similar reversal of IFN-gamma and IL-4 production accompanied progressive disease induced by pretreatment with a single dose of anti-IFN-gamma mAb. Although IFN-gamma production was suppressed for up to 4 wk in mice treated with monoclonal anti-IL-12 or anti-IFN-gamma, coadministration of neutralizing anti-IL-4 IgG reversed progressive illness. These findings demonstrate that IL-12 produced in vivo is necessary for both the emergence of IFN-gamma producing cells and the down-regulation of Th2 cell responses during murine leishmaniasis. Furthermore, the uninhibited production of IL-4 was required to sustain progressive infection initiated by the decreased IFN-gamma synthesis observed in anti-IL-12 and anti-IFN-gamma-treated mice.

Host response to Sendai virus in mice lacking class II major histocompatibility complex glycoproteins.

The development of Sendai virus-specific cytotoxic T-lymphocyte (CTL) effectors and precursors (CTLp) has been compared for mice that are homozygous (-/-) for a disruption of the H-2I-Ab class II major histocompatibility complex glycoprotein and for normal (+/+) controls. The generation of CD8+ CTLp was not diminished in the -/- mice, though they failed to make virus-specific immunoglobulin G class antibodies. While the cellularity of the regional lymph nodes was decreased, the inflammatory process assayed by bronchoalveolar lavage (BAL) of the pneumonic lung was not modified, and potent CTL effectors were present in BAL populations recovered from both groups at day 10 after infection. There was little effect on virus clearance. Production of interleukin-2 by both freshly isolated BAL inflammatory cells and cultured lymph node cells was greatly diminished, though the -/- mice still made substantial levels of gamma interferon. However, treating the mice with a single dose of a monoclonal antibody to this cytokine, at least some of which is made by CD8+ T cells, did not decrease CTLp frequencies. As found previously with CD4-depleted H-2b mice, the development of Sendai virus-specific CD8+ T-cell-mediated immunity is not compromised by the absence of a concurrent class II major histocompatibility complex-restricted response.

Comparison of the T helper cell response induced by respiratory syncytial virus and its fusion protein in BALB/c mice

Specific proliferative T-cell responses were induced in the lymph node cells (LNC) of mice immunised with a sucrose density gradient purified preparation of respiratory syncytial (RS) virus or an immunoaffinity purified preparation of the F glycoprotein. Inhibition studies and flow cytometric analysis showed that the responding cell population were CD4 + T cells. The cytokines produced by virus-specific and F-specific cells were assessed using the CTLL cell line. Peak quantities of cytokine were consistently detected in the supernatants of stimulated cultures 24 h prior to maximum proliferation. The proportion of IL-2 released was determined by blocking IL-2 activity with an anti-IL-2 monoclonal antibody. In cultures of RS virus primed LNC challenged with whole virus there was a switch of cytokine production from 70% IL-2 at day 3 to 80% IL-4 by 6 days of culture. In contrast, LNC cultures from mice immunised with F protein secreted 75–100% IL-2 throughout the culture period. These data suggest that after 6 days of challenge with viral antigen, the RS virus-primed LNC response consists of T helper cells which are predominantly of the Th2 subset, secreting IL-4, whilst F protein-primed LNC secrete large quantities of IL-2 and can therefore be classified as predominantly of the Th1 subset.

Concanavalin A—induced T-cell—Mediated hepatic injury in mice: The role of tumor necrosis factor

Concanavalin A activates T lymphocytes in vitro and causes T-cell-dependent hepatic injury in mice. T lymphocytes were previously identified as effector cells of concanavalin A—induced liver injury. Here we report that hepatic injury is characterized by apoptotic cell death. On concanavalin A challenge, the cytokines tumor necrosis factor-α (TNFα), interleukin-2, granulocyte macrophage-colony stimulating factor, and interferon-γ were detectable in the circulation of the mice. Pretreatment of mice with anti-mouse TNF-α antiserum protected them from concanavalin A-induced liver injury. Nude mice failed to release TNF-α or interleukin-2 after concanavalin A challenge and were protected from liver injury. Lymph node cell transfer from responder mice to resistant nude mice resulted in susceptibility of the latter towards concanavalin A, i.e., to induction of cytokine release and hepatotoxicity. These experiments suggest that immunocompetent T cells play a pivotal role in concanavalin A—stimulated TNF-α release in vivo. After intravenous administration of fluorescein isothiocyanate-labeled concanavalin A to mice, the most fluorescence was found within the liver. In vitro, concanavalin A stimulation of separate cultures of mouse lymph node cells or nonparenchymal liver cells induced the release of minute amounts of TNF, whereas stimulation of cocultures of these cells resulted in production of substantial amounts of TNF-α. These findings may explain the hepatotropic effect of concanavalin A. In conclusion, T-cell-dependent concanavalin A-induced apoptotic liver injury in mice is related to immunological and cytokinemediated disorders and possibly to autoreactive hepatic processes.

Concanavalin A—induced T-cell—mediated hepatic injury in mice: The role of tumor necrosis factor

Concanavalin A activates T lymphocytes in vitro and causes T-cell-dependent hepatic injury in mice. T lymphocytes were previously identified as effector cells of concanavalin A-induced liver injury. Here we report that hepatic injury is characterized by apoptotic cell death. On concanavalin A challenge, the cytokines tumor necrosis factor-α (TNF α), interleukin-2, granulocyte macrophage-colony stimulating factor, and interferon-γ were detectable in the circulation of the mice. Pretreatment of mice with anti-mouse TNF-α antiserum protected them from concanavalin A-induced liver injury. Nude mice failed to release TNF-α or interleukin-2 after concanavalin A challenge and were protected from liver injury. Lymph node cell transfer from responder mice to resistant nude mice resulted in susceptibility of the latter towards concanavalin A, i.e., to induction of cytokine release and hepatotoxicity. These experiments suggest that immunocompetent T cells play a pivotal role in concanavalin A-stimulated TNF-α release in vivo. After intravenous administration of fluorescein isothiocyanate-labeled concanavalin A to mice, the most fluorescence was found within the liver. In vitro, concanavalin A stimulation of separate cultures of mouse lymph node cells or nonparenchymal liver cells induced the release of minute amounts of TNF, whereas stimulation of cocultures of these cells resulted in production of substantial amounts of TNF-α. These findings may explain the hepatotropic effect of concanavalin A. In conclusion, T-cell-dependent concanavalin A-induced apoptotic liver injury in mice is related to immunological and cytokinemediated disorders and possibly to autoreactive hepatic processes. (Hepatology 1995;21:190-198).

Lymphokine‐activated killer cell function of lymphocytes from regional lymph nodes in patients with gastric carcinoma

Lymphokine-activated killer (LAK) cells generated by culture of regional lymph node cells (LNC) with interleukin 2 (IL 2) for 4 and 11 days were examined for their functional capabilities in comparison with those of peripheral blood mononuclear cells (PBM) in 25 patients with gastric carcinoma. The cytotoxic activity of LAK cells induced from LNC for 4-day culture with IL 2 was significantly lower than that from PBM. However, the LNC-LAK cytotoxicity was markedly increased up to almost the same level as that of PBM after 11-day culture. The production of interferon-gamma (INF-gamma) and tumor necrosis factor-alpha (TNF-alpha) from nonadherent LAK cells in LNC was also significantly reduced as compared to that from PBM 4 days after culture, when stimulated with or without tumor target, Raji cells. After 11-day culture with IL 2, however, the levels of these cytokines produced by LNC-LAK cells either with or without stimulation by tumor target were comparable to those by PBM-LAK cells, although the release of these cytokines was markedly reduced when compared to that after 4-day culture. Phenotypic analysis revealed decreased proportion of cells mediating NK activity in LNC before and 4 days after culture. CD56+ and CD57+ cells in LNC were increased after 11-day culture, although the percentages of these cells were still low as compared to those in PBM. The proportions of OKIa1+ and CD25+ cells were uniformly increased after 4 and 11-day culture in both cell populations. Changes in subpopulations of CD4+ and CD8+ cells in LNC were not apparently different from PBM. These results indicated the differential LAK cell function of cells from regional lymph nodes from PBM in patients with gastric carcinoma.

Immunoregulatory properties of hexapeptide isolated from porcine bone marrow cell culture

Myelopeptide 1 (MP-1) is hexapeptide originally isolated from porcine bone marrow cell culture. It was synthesized and its immunoregulatory properties were studied. MP-1 caused a 1.5–2-fold dose-dependent increase of antibody production in the culture of mouse immune lymph node cells. It abolished Con A induction of T suppressors in the suspension of mouse spleen cells and counteracted the inhibitory effect of T suppressors on antibody production. The inoculation of MP-1 (1·10 −9 g/mouse) to mice two weeks after their γ-irradiation (2 Gy) resulted in an increase of antibody production up to 80.2 ± 15.5% as compared to that in the irradiated control 37.6 ± 12.0%. Immunofluorescent analysis revealed the specific binding of MP-1 with receptors on the target cells in the suspension of mouse spleen cells. It is supposed that MP-1 participates in the immunoregulatory processes in the living organism.

Interleukin 6 production in vitro: An alternative read-out for the local lymph node assay

The murine local lymph node assay has been developed as an alternative method for the identification of contact allergens. In contrast to guinea pig tests, which rely on visual assessment of challenge-induced dermal reactions, the local lymph node assay measures events occurring during the induction of skin sensitization. Contact allergic potential is measured as a function of hyperplastic responses in draining lymph nodes following systemic administration of [ 3H]thymidine. We have now examined whether the production in vitro of interleukin 6 (IL-6) by draining lymph node cells isolated from sensitized mice provides an alternative endpoint for the local lymph node assay. In comparative experiments, the production of IL-6 by lymph node cells in culture correlated closely with proliferative responses in vitro. Only chemicals known to cause contact sensitization elicited measurable (> 150 pg/ml) IL-6 production; non-sensitizing chemicals, including skin irritants, did not. Experience to date suggests that IL-6 production may provide a useful alternative read-out for the identification of chemicals which have a significant skin-sensitizing potential.