Suppression of footpad reaction in mice by 12-tetradecanoylphorbol 13-acetate and 7,12-dimethylbenz[a]anthracene

The effect of treatment with 12-tetradecanoylphorbol 13-acetate (TPA), a tumor promoter, following 7,12-dimethylbenz[a]anthracene (DMBA), a tumor iniator, on foodpad reaction (FPR) with sheep blood red cells (SRBC) in mice was studied. After painting of 400 nmol of DMBA on the skins of the BALB/c mice immunized with SRBC, 8 nmol of TPA was painted every day for 7 days. Then, FPR was measured 24 hr after 2nd injection of SRBC. In these experiments, FPR was markedly suppressed and this effect continued for 2 weeks. When the spleen cells from the unimmunized donor mice which had been treated with DMBA and TPA were injected into the recipient mice was suppressed. These data show that the suppressive effect on FPR may be caused by the increase of the suppressor cells which are not specific for the antigen. This effect disappeared by the treatment of the spleen cells with Thy-1 or Lyt-2 antibody. These results suggest that the suppressor cells has Thy-1 and Lyt-2 antigens.     

CELLULAR RECOGNITION BY MOUSE LYMPHOCYTES IN VITRO : I. DEFINITION OF A NEW TECHNIQUE AND RESULTS OF STIMULATION BY PHYTOHEMAGGLUTININ AND SPECIFIC ANTIGENS

The media and culture conditions required for in vitro stimulation of mouse lymphoid cells are described. The medium was arginine-rich and contained heat-inactivated human serum. A component of the human sera necessary for stimulation of the cells was a natural mouse cell agglutinin, which affected both background stimulation and the degree of induced stimulation with phytohemagglutinin (PHA). Absorption of the agglutinin from the human serum rendered the medium incapable of sustaining DNA synthesis in the presence of PHA. The response to PHA of mouse spleen and thymus cells was age-dependent and, although this response was not present at birth, it rapidly rose to adult levels. Spleen cells from mice immunized with bacillus Calmette-Guérin (BCG) or sheep erythrocytes (SRBC) showed increased in vitro reactivity to added purified protein derivative (PPD) or SRBC stroma, dependent on the time of immunization. The dose response curve for the SRBC stroma stimulated, immune spleen cells is compatible with a theory of cell to cell interaction being necessary for an in vitro reaction to antigen. The possible role of the mouse cell agglutinin (AMLG) is discussed.     

Local and systemic opsonic adherent, hemagglutinating and rosette forming activity in mice induced by respiratory immunization with sheep red blood cells.

The humoral response in mice after intranasal (IN) instillation of sheep red blood cells (SRBC) was compared to that produced by intraperitoneal route of immunization. Study of opsonic adherent (passive indirect cytophilic) and hemagglutinating antibody levels in serum and bronchial washings after repeated challenge with SRBC showed that serum antibody titers following respiratory immunization does not differ from the general pattern of a humoral antibody response. The bronchial wash antibody titer was higher than the serum titer as calculated per globulin unit. In contrast, the appearance of measurable antibodies in the bronchial washings after repeated intraperitoneal immunization was delayed and in low titer. B. pertussis vaccine administered in conjunction with SRBC was found to be an excellent adjuvant in the intranasal route of immunization. A single administration of SRBC-Pertussis evoked a considerable adjuvant effect as measured by hemagglutinating and opsonic adherent tests. Monospecific anti-mouse IgG nullified the hemagglutinating and opsonic adherent activity of the bronchial washings, while monospecific anti-mouse IgM had no effect in either activity. Lung and spleen cells capable of forming rosettes with SRBC of intranasally immunized and control mice were counted. Mean response on day 14 was 6 X 10(3) rosetes per million lung lymphoid cells in comparison with less than 10(3) in control mice. No difference was found in the number of rosette-forming cells in the spleen of control and intranasally immunized mice.     

THE EFFECT OF HETEROLOGOUS ANTI-LYMPHOCYTE SERUM ON LYMPHOCYTES OF THYMUS AND MARROW ORIGIN

When CY-treated mice were given sheep red blood cells the serum hemagglutinin titers produced were significantly lower than those found when mice received SRBC but not CY. Titers could be raised to the levels found in the latter group if, in addition to SRBC, the CY-treated mice received 2 x 10⁷ normal syngeneic spleen cells or a mixture containing 1.2 x 10⁸ normal thymus and 1.2 x 10⁸ normal marrow cells. Inocula which contained fewer cells produced correspondingly smaller amounts of antibody. A synergistic interaction between normal thymus and marrow cells was always demonstrable in these experiments. Hemagglutinin titers produced by CY-treated mice given SRBC and 2 x 10⁷ normal syngeneic spleen cells were always much higher than those found when the spleen cells were obtained from animals previously given ALS. Titers could be raised to normal levels if the animals in this latter group received additional injections containing mixtures of normal syngeneic thymus and marrow cells. Marrow cells alone were completely ineffective, while inocula which only contained thymus cells were much less effective than mixtures of thymus and marrow cells. These results suggest that immunosuppression by ALS is associated with the inactivation of both thymus and marrow-derived lymphocytes. In other experiments CY-treated mice received SRBC and mixtures of thymus and marrow cells from both untreated and ALS-treated donors. No decrease in the immunological competence of cells located within the thymus of ALS-treated donors was demonstrable in these experiments. Marrow cells were slightly affected but to a markedly lesser degree than were spleen cells of ALS-treated animals. In a final experiment, it was possible to show that the thymus and marrow cells of ALS-treated animals could repair the immunological defects which were present in their own spleen cell populations.     

Thymus-derived rosettes are not "helper" cells

Rosettes against SRBC were made from normal spleen cells. Although T rosettes tend to dissociate, they could be stabilized with 0.05% sodium azide. A clear separation of nonrosettes, T rosettes, and B rosettes was obtained by subjecting the suspension of splenic rosettes to velocity sedimentation at unit gravity. Each fraction was injected with either normal bone marrow cells or normal thymus cells with antigen into 650-R-irradiated hosts. Direct plaque-forming cells (PFC) were assayed in the spleens 7 days later. Synergism with thymus cells occurred only in the B-rosette fraction; PFC precursors therefore sedimented as B rosettes. Synergism with bone marrow cells occurred only in the nonrosette small lymphocyte fraction; helper cells therefore did not bind detectable numbers of sheep red blood cells (SRBC). Thus T rosettes are not helper cells in the direct PFC response of bone marrow B cells to SRBC.     

Antigen specific receptors on murine B cell lineage. 1. On antibody forming cells in various stages of immune response

In this study it was examined whether or not antigen specific receptors are on direct and indirect plaque-forming cells (PFC). Spleen cells from mice primed both with horse red blood cells (HRBC) and sheep red blood cells (SRBC) were incubated with HRBC to make rosettes and fractionated on Ficoll-Hypaque density gradient. Both direct and indirect HRBC-PFC were depleted from the interface fraction. The depletion was antigen specific and inhibited by the preincubation of spleen cells with antiserum against kappa- or mu-chain for direct PFC and with antiserum against kappa- or gamma 1-chain for indirect PFC. The depletion was not due to the rosette formation mediated by Fc-receptors which might be on antibody forming cells, because PFC were not eliminated by the density gradient sedimentation after incubation of spleen cells with SRBC coated with anti-SRBC IgG antibody in our experimental conditions. Our results show that the antigen specific immunoglobulin receptors are on the cells producing IgG antibody after primary or secondary immunization as well as on the IgM antibody-forming cells even in late stage of immune response.     

Augmented delayed footpad reaction in thymus cell-depleted mice induced by cholera toxoid

One hundred microgram of cholera toxoid was injected intravenously into DDD and AKR mice and its effects on lymphoid tissues and immune responses against sheep erythrocytes (SRBC) were examined at various times after the injection. (1) A remarkable reduction of thymus cells was revealed from day 1 to 7 and from day 1 to 4 in DDD and AKR mice, respectively. (2) Cholera toxoid exhibited only slight effects on the numbers of spleen cells and peripheral blood leukocytes in both strains. (3) Delayed footpad reactions to SRBC were augmented by a pretreatment with cholera toxoid 4 or 7 days before immunization in both strains. The delayed reactions were not suppressed in the presence of a prominent antibody production and were accompanied by positive macrophage migration inhibition. (4) Antibody production against SRBC, especially of IgG class, was facilitated, when cholera toxoid was given 7 days before the immunization through the footpad in DDD mice. On the other hand, antibody production was suppressed irrespective of immunizing routes and mouse strains, when cholera toxoid was given 1 day before immunization.     

不同动物红细胞制备实验教学用溶血素的方法探讨

目的检测绵羊红细胞(SRBC)和猪红细胞(PRBC)分别经2种程序免疫家兔所制得溶血素的效价,并比较几种溶血素在医学免疫学实验中的应用效果,以寻求制备符合免疫学实验及教学需求的高效价溶血素的较好方法。方法 40只雄性家兔分为4组,分别用SRBC和PRBC,间隔2d经不同程序免疫家兔制备溶血素,再用补体溶血试验检测几种溶血素的效价,比较其在免疫学实验教学中的应用,并探讨高效价溶血素的制备方法。结果经补体溶血试验测得几种溶血素效价,A组兔抗-SRBC血清效价为1∶4 800;B组兔抗-PRBC血清效价为1∶1 200;C组兔抗-SRBC血清效价为1∶1 000,D组兔抗-PRBC血清效价为1∶200。结论用SRBC制备的溶血素效价均高于相同方法下用PRBC制备的溶血素;红细胞抗原相同时,采取先皮内全血注射后耳缘静脉接种的免疫方法制备的溶血素效价也要高于单纯采取耳缘静脉注射的免疫方法制备的溶血素;且PRBC可代替SRBC免疫家兔,制备满足实验教学要求的溶血素。     

CELLS INVOLVED IN THE IMMUNE RESPONSE : VI. THE IMMUNE RESPONSE TO RED BLOOD CELLS IN IRRADIATED RABBITS AFTER ADMINISTRATION OF NORMAL, PRIMED, OR IMMUNE ALLOGENEIC RABBIT BONE MARROW CELLS

Irradiated rabbits given allogeneic bone marrow cells from normal adult donors responded to an injection of sheep red blood cells by forming circulating antibodies. Their spleen cells were also capable of forming many plaques using the hemolysis in gel technique, and were also capable of undergoing blastogenesis and mitosis and of incorporating tritiated thymidine upon exposure to the specific antigen in vitro. However, irradiated rabbits injected with allogeneic bone marrow obtained from rabbits injected with sheep red blood cells 24 hr prior to sacrifice (primed donors) were incapable of mounting an immune response after stimulation with sheep red cells. This loss of reactivity by the bone marrow from primed donors is specific for the antigen injected, since the immune response of the irradiated recipients to a non-cross-reacting antigen, the horse red blood cell, is unimpaired. Treatment of the bone marrow donors with high-titered specific antiserum to sheep red cells for 24 hr prior to sacrifice did not result in any diminished ability of their bone marrow cells to transfer antibody-forming capacity to sheep red blood cells. The significance of these results, with respect to the origin of the antigen-reactive and antibody-forming cells in the rabbit, is discussed.     

THE EFFECTS OF ANTIGENS AND OF PHYTOHEMAGGLUTININ ON RABBIT SPLEEN CELL SUSPENSIONS

Phytohemagglutinin (PHA) stimulated the rate of DNA synthesis in rabbit spleen cell suspensions. Unlike antigens, previous immunization to PHA was not necessary and the specific response could not be transferred by macrophages, although lymphocytes primed by incubation in PHA were able to stimulate other spleen cells not directly exposed to PHA. When rabbits were stimulated by in vivo immunization with antigens, spleen cells proliferating in response to antigen were stimulated to divide by in vitro contact with PHA. Using the technique of specific hemolytic plaque formation by individual cells synthesizing γM-antibody to sheep red cells (plaque-forming cells), no evidence was obtained that stimulation of cell division by PHA resulted in specific antibody formation, although the presence of antigen resulted both in stimulation of cell proliferation and the production of plaque-forming cells. The presence of both sheep red cells and PHA in the medium of the same cell suspensions did not enhance the production of plaque-forming cells although there was a summative effect on DNA synthesis.     

Virus-replicating T cells in the immune response of mice. I. Virus plaque assay of the lymphocytes reactive to sheep erythrocytes

Virus plaque-forming cell assay with vesicular stomatitis virus (VSV), which had been originally introduced by Bloom and his colleagues as a tool for the enumeration of activated lymphocytes, was first applied to the immune response of mice to a widely used antigen, i.e. sheep red blood cells (SRBC). When spleen cells taken from mice previously primed with SRBC were cultured in the presence of the antigen, lymphocytes capable of replicating VSV (antigen-induced virus plaque-forming cells, Ag-V-PFC) were generated in the culture. They seemed to appear as early as 1 day of culture, and the peak was attained by the 2nd day. Most of Ag-V-PFC belonged to T-cell population, since 80-90% of Ag-V-PFC was killed by the treatment of cultured cells with anti-thymocyte serum plus complement. In vitro generation of Ag-V-PFC seemed to be highly cross-reactive (about 40%) with a related antigen (horse red blood cells). Ag-V-PFC detected in the present experiment may not represent helper T cells, effector T cells, or their precursors because of the following: (a) The generation of Ag-V-PFC was completely suppressed by the addition of anti-SRBC mouse serum in the culture, though the helper activity was apparently augmented by the same treatment. (b) Development of Ag-V-PFC was almost completely suppressed by the pretreatment of mice with cyclophosphamide 2 days before immunization, by which delayed-type hypersensitivity (DTH) was markedly augmented. (c) After the immunization of mice, Ag-V-PFC began to develop just when the level of DTH declined, at which point helper activity of the spleen cells also diminished. A possible role of Ag-V-PFC in the immune response was discussed.     

INFLUENCE OF DOSE AND ROUTE OF ANTIGEN INJECTION ON THE IMMUNOLOGICAL INDUCTION OF T CELLS

Delayed-type hypersensitivity (DTH) develops in the absence of an adjuvant when mice are injected intravenously or subcutaneously with an appropriate dose of sheep red blood cells (SRBC). The optimal intravenous dose of 10⁵ SRBC (in CD-1 mice) produces maximum DTH which decays exponentially from its peak on day 4. Increasing the dose of SRBC reduces and eventually abolishes all evidence of DTH. DTH fails to reappear in respose to secondary stimulation except in splenectomized mice in whom the development of DTH is not suppressed, even by massive doses of SRBC. Hence the suppression cannot be due to antigen as such. The optimal dose of SRBC for sensitization by footpad inoculation is 100-fold higher (10⁷ SRBC in CD-1 mice), but even 10⁹ SRBC do not block the induction of DTH by this route of immunization. A blocking dose of SRBC, given intravenously 1 day before footpad inoculation, completely suppresses cell proliferation in the draining lymph node, prevents PFC production there, and blocks the induction of DTH by a sensitizing dose of SRBC. If given 1 day after footpad sensitization, intravenous antigen has little effect on the cellular response in the regional node but DTH is still completely suppressed. Blocking of induction and expression may depend, therefore, on different mechanisms.     

PRIMARY ANTIBODY RESPONSE IN VITRO IN PERITONEAL CELLS

Peritoneal cells (PC), from nonimmunized mice, incubated in a carboxymethyl cellulose gum containing sheep red blood cells (SRBC) and guinea pig complement (following the technique of Ingraham and Bussard) start to produce plaques of hemolysis 20 hr after the beginning of incubation at 37°C. In contrast, spleen cells from immunized mice complete their plaque-forming activity in 6 hr. The fact that formation of plaques by previously uncommitted cells is related to the life of the leukocytes, and is complement dependent brings evidence that we are dealing with an immunological phenomenon. Puromycin suppresses the formation of plaque. Previous incubation of the PC with SRBC in liquid medium, before incorporation in the detection system, reduces the lag in the production of plaques. This indicates that a phase of stimulation precedes the phase of expression (manifested by plaque formation). The immunological activity of the peritoneal cell suspension is highly dependent on the concentration of the suspension, which indicates that this activity results from a cooperative process. Actinomycin D, which does not suppress the production of plaques by cells from immunized animals, stops completely the in vitro induction. It is concluded that we have probably observed a primary immune response induced in vitro.     

TONE AND REACTIVITY OF VASCULAR SMOOTH MUSCLE IN GERMFREE RAT MESENTERY

The immune responses to sheep erythrocytes of mouse spleen cell suspensions from immune and nonimmune donors were compared in vitro. In vivo immunity was only transiently reflected in vitro, and 8 wk after in vivo immunization the responses of cultures from immunized and nonimmunized mice were virtually identical. There appeared to be two mechanisms for an antibody response to sheep erythrocytes. The first was responsible for the early primary response and is unmodified in the immune animal though contributing little to subsequent in vivo responses due to its suppressibility by specific antibody. The second was expressed in the in vivo secondary response but not on in vitro challenge of spleen cells from mice immunized many weeks previously; spleen cell cultures from such immune mice, freed from the antibody of the in vivo environment, once again demonstrate a pure primary-type response.     

REGULATION OF CELLULAR ANTIBODY SYNTHESIS : CELLULAR 7S PRODUCTION AND LONGEVITY OF 7S ANTIGEN-SENSITIVE CELLS IN THE ABSENCE OF ANTIBODY FEEDBACK

Transfer of spleen cells from mice immunized against sheep red blood cells (SRBC) into irradiated (600 R) nonimmune, syngeneic mice in the presence of antigen resulted in excessive cellular 7S production 7 days later. The number of 7S plaque-forming cells usually exceeded 10⁶ per spleen and the mean proportion varied between 1 and 70%. In occasional animals all spleen cells were producing antibodies to SRBC. Serum antibody synthesis was also excessively increased, the titers in agglutination after 2-ME treatment and in hemolysis varying between 2¹⁵ and 2²⁵. The generation time of the 7S PFC was found to be 9.6 hr in the secondary hosts. It seemed possible that the excessive production of 7S PFC and antibodies in the irradiated nonimmune recipients was caused by the absence of feedback inhibition of the immune response by antibody, a mechanism which would normally function to restrict antibody synthesis. This conclusion was strengthened by the demonstration that transfer of antigen-stimulated immune cells into actively or passively immunized irradiated recipients resulted in a marked suppression of cellular 7S synthesis. Serial transfers of antigen-stimulated immune cell populations in irradiated hosts resulted in an equally high number of 7S PFC during the first four transfer generations. However, after the fifth to seventh transfer generation the number of 7S PFC rapidly declined and disappeared within one to three passages. Serum antibodies and 7S PFC declined in parallel during the last transfer generations. Further passages of antigen-stimulated spleen cells lacking 7S PFC did not lead to reappearance of PFC. Thus, antigen-sensitive cells have a limited lifespan and/or multiplication capacity. From the hypothesis that the 7S PFC developed by division from antigen-sensitive precursors it was calculated that 38–40 divisions occurred, Thus, one antigen-sensitive precursor has the potential to give rise to 10¹² 7S PFC.     

CELL TO CELL INTERACTION IN THE IMMUNE RESPONSE : V. TARGET CELLS FOR TOLERANCE INDUCTION

Collaboration between thymus-derived lymphocytes, and nonthymus-derived antibody-forming cell precursors occurs during the immune response of mice to sheep erythrocytes (SRBC). The aim of the experiments reported here was to attempt to induce tolerance in each of the two cell populations to determine which cell type dictates the specificity of the response. Adult mice were rendered specifically tolerant to SRBC by treatment with one large dose of SRBC followed by cyclophosphamide. Attempts to restore to normal their anti-SRBC response by injecting lymphoid cells from various sources were unsuccessful. A slight increase in the response was, however, obtained in recipients of thymus or thoracic duct lymphocytes and a more substantial increase in recipients of spleen cells or of a mixture of thymus or thoracic duct cells and normal marrow or spleen cells from thymectomized donors. Thymus cells from tolerant mice were as effective as thymus cells from normal or cyclophosphamide-treated controls in enabling neonatally thymectomized recipients to respond to SRBC and in collaborating with normal marrow cells to allow a response to SRBC in irradiated mice. Tolerance was thus not achieved at the level of thelymphocyte population within the thymus, perhaps because of insufficient penetration of the thymus by the antigens concerned. By contrast, thoracic duct lymphocytes from tolerant mice failed to restore to normal the response of neonatally thymectomized recipients to SRBC. Tolerance is thus a property that can be linked specifically to thymus-derived cells as they exist in the mobile pool of recirculating lymphocytes outside the thymus. Thymus-derived cells are thus considered capable of recognizing and specifically reacting with antigenic determinants. Marrow cells from tolerant mice were as effective as marrow cells from cyclophosphamide-treated or normal controls in collaborating with normal thymus cells to allow a response to SRBC in irradiated recipients. When a mixture of thymus or thoracic duct cells and lymph node cells was given to irradiated mice, the response to SRBC was essentially the same whether the lymph node cells were derived from tolerant donors or from thymectomized irradiated, marrow-protected donors. Attempts to induce tolerance to SRBC in adult thymectomized, irradiated mice 3–4 wk after marrow protection, by treatment with SRBC and cyclophosphamide, were unsuccessful: after injection of thoracic duct cells, a vigorous response to SRBC occurred. The magnitude of the response was the same whether or not thymus cells had been given prior to the tolerization regime. The various experimental designs have thus failed to demonstrate specific tolerance in the nonthymus-derived lymphocyte population. Several alternative possibilities were discussed. Perhaps such a population does not contain cells capable of dictating the specificity of the response. This was considered unlikely. Alternatively, tolerance may have been achieved but soon masked by a rapid, thymus-independent, differentiation of marrow-derived lymphoid stem cells. On the other hand, tolerance may not have occurred simply because the induction of tolerance, like the induction of antibody formation, requires the collaboration of thymus-derived cells. Finally, tolerance in the nonthymus-derived cell population may never be achieved because the SRBC-cyclophosphamide regime specifically eliminates thymus-derived cells leaving the antibody-forming cell precursors intact but unable to react with antigen as there are no thymus-derived cells with which to interact.     

ANTIBODY AND IMMUNOGLOBULIN PRODUCTION AT THE CELLULAR LEVEL IN BURSECTOMIZED-IRRADIATED CHICKENS

The effect of bursectomy combined with sublethal X-irradiation in the newly hatched chicken on the immunoglobulin and antibody producing capacity in later life was investigated. The previous findings of a significant incidence of hypogammaglobulinemia in such animals were confirmed. Spleen cells from severely hypogammaglobulinemic animals synthesized and secreted little or no immunoglobulin. Such spleen lymphoid cells contained fewer immunoglobulin antigenic determinants than spleen cells from irradiated control animals as evidenced by their relative inability to respond by an increased DNA synthesis after in vitro culture with rabbit antiserum to chicken immunoglobulin. Therefore, the deficiency in the immunoglobulin synthesis extends not only to actively secreting cells such as plasma cells, but to the entire lymphoid cell population. As expected, most irradiated-bursectomized chickens, irrespective of plasma immunoglobulin levels failed to produce detectable amount of circulating antibodies to Brucella abortus antigen in the primary immune response. Severely hypogammaglobulinemic animals were completely unable to elaborate any plaque forming cells (PFC) in the primary response to sheep red blood cells (SRBC). The results of this investigation support the contention that in the severely hypogammaglobulinemic bursectomized-irradiated chicken the entire antibody producing and immunoglobulin producing cell line is absent. The possibility remains that precursor or stem cells are present but are not appropriately directed to antibody synthesis by other cell types.     

EFFECT OF GROWTH HORMONE ON THE METABOLISM OF THYMUS AND ON THE IMMUNE RESPONSE AGAINST SHEEP ERYTHROCYTES

The effect of pituitary growth hormone on the biosynthesis of DNA in the thymus and other lymphoid organs, as well as the ability of the rat to respond immunologically to sheep red blood cells, has been evaluated. There is a marked reduction in plaque-forming cells, hemagglutination titers, and DNA synthesis in animals when examined at 15 wk after hypophysectomy. Administration of bovine growth hormone (BGH) leads to the enhancement of DNA synthesis in lymphoid organs and recovery of the immune response. Similar effects of the hormone are observed in plateaued rats. Injection of rabbit anti-BGH globulins, in contrast to normal rabbit globulins, over 5 days causes a drop in the weight of the thymus and in the rate of DNA synthesis in this organ. The thymus is also the organ in which stimulation of DNA synthesis is observed at a time period earlier than the spleen and lymph nodes after a single injection of BGH. The hormone stimulates not only the incorporation of thymidine-³H into DNA in the cortical cells, but also the incorporation of sodium sulfate-³⁵S into TCA-insoluble biopolymers reported to be elaborated in the medullary area of the thymus. An in vitro system for the action of BGH on the thymus has been described. There is an obligatory requirement for calcium, but not for fetal calf serum in the medium for the hormone effect. An early action of the hormone is the enhanced incorporation of uridine-G-³H into RNA in thymocytes which is followed by a stimulation of the synthesis of proteins and DNA. The stimulatory action of growth hormone on RNA synthesis is not because of a facilitated uptake of the radioactive uridine by the cells under hormonal influence, a mechanism by which insulin is observed to increase RNA synthesis in thymocytes in vitro. The action of growth hormone on thymocytes is specific, since thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and heat-inactivated growth hormone are not effective. BGH has also a beneficial action on the regeneration of the thymus and spleen in starved rats.     

Mechanism of suppression of footpad reaction in mice by 12-tetradecanoylphorbol 13-acetate and 7,12-dimethylbenz[a]anthracene

We reported previously that footpad reaction (FPR) of BALB/c mice against sheep red blood cells (SRBC) was suppressed by the percutaneal administration of 12-tetradecanoyl 13-acetate (TPA), a tumor promoter, following that of 7,12-dimethylbenz[a]anthracene (DMBA), a tumor initiator. This effect could be transferred with Thy-1(+) and Lyt-2(+) spleen cells. These findings suggested that this effect was caused by induction of the antigen nonspecific T-suppressor cells and that the process of the induction consisted of two steps. In the present report, we studied the cells related to the first step of this process. The spleen cells from the donor mice on which 400 nmol of DMBA was painted were transferred to the recipient mice which had been immunized 1 hr before the transfer. Then, 8 nmol of TPA was painted on the recipient mice every day for a week. Nine days after the transfer, FR against SRBC was measured. FPR in the recipient mice was suppressed compared to the control, showing that the effect of DMBA can be transferred with the spleen cells. The results of the treatment of the cells to be transferred with monoclonal antibodies and complement show that the cells which is responsible for the transfer of the effect are Mac-1(+), Thy-1(-), Lyt-2(-) and Ia(-). Also the effect of DMBA could be transferred with peritoneal macrophages from the donor mice on which DMBA was painted and anti Ia antibody treatment of the macrophages did not abrogate the effect of transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction and abrogation of unresponsiveness in nude mouse cells

Nude mice were injected with antigen and T cells at different times to induce unresponsiveness to SRBC. Spleen cells derived from these mice were tested in vitro for the capability to produce antibody-forming cells against sheep erythrocytes in the presence of a T-cell-replacing factor. It was found that priming with antigen alone did not result in paralysis but a later injection of thymus-derived lymphocytes together with antigen results in unresponsiveness of these cells in vitro, provided there was an interval of several days between the in vivo administration of thymus lymphocytes and the explantations of cells to in vitro cultures.