Dual effects of OK-432 on mitogenic response of splenocytes to concanavalin A

OK-432, a streptococcal preparation, was studied for its effect on the concanavalin A (Con A)-induced mitogenesis of the host spleen cells. When mice were given a single intraperitoneal injection of OK-432, there was a substantial increase in the mitogenic response of splenocytes, whereas multiple injections conversely resulted in a marked reduction of the mitogenic response, when the spleen cells were cultured at high cell densities of over than 5 X 10(5) cells/well. The reduced Con A-responsiveness in the latter was not restored by mixing spleen cells from mice given multiple OK-432 injections with those from normal mice. Moreover, splenic macrophages from OK-432-injected mice exhibited marked inhibitory activity against Con A-mitogenesis of normal splenocytes, while normal splenic macrophages failed to show such an effect. Splenic T cells from OK-432-injected mice also showed an inhibitory activity against Con A-mitogenesis of normal splenocytes and similar activity was also noted in normal splenic T cells. Therefore, the OK-432-spleen cells contain two types of suppressor cells; one is a newly elicited suppressor macrophage and the other is a suppressor T cell supposedly resident also in normal spleen cells. In the OK-432-injected spleen cells, accessory cell function for T cell Con A-mitogenesis was markedly reduced. On the other hand, it was noted that the interleukin 2-producing ability of the OK-432-splenocytes was augmented more than that of normal splenocytes, indicating that multiple OK-432 injections also cause an increase in the helper T cell activity of the host spleen cells.     

Elevation of glutathione induced by low-dose gamma rays and its involvement in increased natural killer activity

We examined the relationship between the induction of an increase in the level of glutathione and the elevation of natural killer (NK) activity in mouse splenocytes by a low dose of gamma rays. The glutathione levels in mouse splenocytes increased significantly between 2 h and 6 h after whole-body gamma irradiation at 0.5 Gy, peaked at 4 h, and then decreased almost to the level before irradiation by 12 h postirradiation. A significant enhancement of NK activity was found in the splenocytes obtained from whole-body-irradiated mice between 4 and 6 h postirradiation. Reduced glutathione (GSH) added exogenously to splenocytes obtained from normal mice enhanced both the total cellular glutathione content and the NK activity in a dose-dependent manner. Other precursors of de novo GSH synthesis, such as cysteine, N-acetylcysteine and oxidized glutathione, also increased the activity. These enhancements were completely blocked by buthionine sulfoximine, an inhibitor of de novo GSH synthesis. We conclude that the induction of endogenous glutathione in living cells immediately after low-dose gamma irradiation is at least partially responsible for the appearance of enhanced NK activity.     

Depletion of Dendritic Cells Enhances Innate Anti-Bacterial Host Defense through Modulation of Phagocyte Homeostasis

Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection.     

Effect of synthetic beta-carotene on the formation of cytolytic T-lymphocytes

The effect of intraperitoneal injection of beta-carotene in different doses on the formation of cytolytic T lymphocytes (CTL) in a one-way mixed lymphocyte culture (MLC) of allogeneic mice was studied. The maximal cytotoxic activity of lymphocytes was attained in the MLC with splenocytes of mice which received 10 mg/kg beta-carotene 6 days before experimentation. The correlation was studied between the beta-carotene ability to stimulate CTL formation and antineoplastic activity. It was discovered that injection of beta-carotene in doses and times provoking maximal CTL induction had no effect on the animals' lifespan and the size of transplanted sarcoma 180.     

Splenocytes from NZB mice exhibit spontaneously high rates of fusion compared to control strains

A previous report (G. E. Woloschak and D. Senitzer, submitted for publication) has demonstrated that mitogen-stimulated splenocytes fuse at a much higher frequency than untreated splenocytes as measured by the fusion index (a calculation of the number of nuclei in fused cells vs the total number of nuclei). A measurement of the fusion indices of NZB spleen cells provides results markedly different from those observed with splenocytes from control strains of mice—NZB spleen cells exhibit a spontaneously high fusion index. In this assay, they spontaneously display the same fusing capacity as that observed in cultures of mitogen-treated spleen cells from control strains of mice. This elevated fusion index is not affected by treatment with anti-Thy-1.2 and complement, but is abrogated by treatment with anti-immunoglobulin serum and complement. This suggests that a B cell is responsible for the high fusion index of NZB splenocytes. This high fusion index is present when using splenocytes from both male and female mice in the fusion assay, and can be observed using spleen cells from NZB mice as young as 12 days. This appears to be the result of a spontaneous polyclonal B-cell activation in NZB mice.     

Interleukin 2 deficiency in murine Leishmaniasis donovani and its relationship to depressed spleen cell responses to phytohemagglutinin

This study examined the kinetics and mechanisms of depressed spleen cell responses to phytohemagglutinin (PHA) that occur during Leishmania donovani infection of BALB/c mice. In co-culture experiments, neither spleen cells from infected animals nor parasite-infected macrophages suppressed PHA responses of normal spleen cells. In addition, parasite-mediated suppression of PHA-stimulated spleen cell proliferation could not be demonstrated. Mice with 2 wk of infection did manifest an impairment in spleen cell production of interleukin 2 (IL 2) and by 8 wk IL 2 activity in supernatants from these cells was reduced by approximately 95%. This finding was not explained by an alteration in the kinetics of IL 2 production. Furthermore, diminished IL 2 activity in supernatants of PHA-activated spleen cells from infected animals was not caused by suppressive factors in these fluids as shown by their inability to suppress IL 2 stimulation of IL 2-dependent T cells. When spleen cells from mice with 8 wk of infection were cultured with PHA and supplemented with exogenous IL 2, there was an approximately 48% increase in mitogenesis. These data indicate that abnormal PHA-induced spleen cell activation in BALB/c mice with L. donovani infection is associated with impaired production of IL 2. In addition, the observation that supplementation of spleen cells from infected mice with IL 2 resulted in partial reconstitution of the PHA response is consistent with a defect in IL 2 responsiveness.     

Activation of T cells in tumor-bearing mice

Subcutaneous transplantation of the syngeneic P815 mastocytoma in DBA/2J mice induced an activation of splenic T cells which resulted in a hyperresponsiveness of the tumor-bearing animal to the unrelated antigens pneumococcal polysaccharide (Pn) and sheep red blood cells (SRBC). These tumor-activated T cells appeared to increase the plaque-forming cell (PFC) potential of suboptimal numbers of spleen cells, caused normal spleen cells to express increased numbers of PFC, and produced lymphokine(s) which also increased PFC responses of normal splenocytes. The tumor-activated T cells responsible for stimulating normal splenocytes in an in vitro antibody response were shown to be Ly+2- cells. The activity of the tumor-activated T-cell supernatants was not genetically restricted and required additional Ly1 T cells in order to induce rigorously clean B cells to produce antibody. The T cells capable of stimulating non-specific antibody responses were also capable of slowing tumor growth when injected with tumor cells in normal recipient mice. These results suggest that T cells activated by tumor antigens release immunostimulatory lymphokines and, at the same time, are capable of leading to inhibition of tumor growth.     

Glutathione status during the mitogenic response of rat splenocytes. Effects of oxygen concentration: FO2 21% versus FO2 7%

Glutathione is known to be an important parameter for ConA proliferative response of murine splenocytes. We studied the glutathione status of ConA-stimulated rat splenocytes during the early and late phase of the mitogenic response under low (FO2 7%) and standard (FO2 21%) oxygen concentrations. We determined the intracellular total, oxidized and reduced glutathione levels after 6, 12, 24 and 48 h of culture with or without ConA and/or 2-ME, under FO2 7% and 21%. Our results showed that: The 2 GSSG/GSH + 2 GSSG ratio, which indicated the redox state of the cells, remained normal during the early period of culture (0-24 h), irrespective of culture conditions. After 48 h of culture, this ratio increased dramatically under FO2 21% and less under FO2 7%. The maintenance of the redox state seems to be an oxygen concentration-dependent phenomenon. ConA stimulation involved a glutathione consumption during the early stages of culture; under these conditions 2-ME increased the glutathione synthesis, which was higher under FO2 7% than under FO2 21%. On the other hand, the presence of 2-ME involved an increase of tritiated thymidine uptake in stimulated splenocytes, which was significantly higher under FO2 21% than under FO2 7%. Low oxygen tension (FO2 7%) can induce a higher increase of glutathione synthesis, whereas the respective ConA proliferative response is lower than that observed under standard O2 conditions.     

Suppressor cell regulation of encephalitogenic T cell lines: generation of suppressor macrophages with cyclosporin A and myelin basic protein.

Chronic relapsing experimental allergic encephalomyelitis (CR-EAE) can be adoptively transferred using myelin basic protein (BP)-specific helper T cell lines, and suppressor cells may be important in recovery from EAE. In order to generate suppressor cells, spleen cells obtained from BP-complete Freund's adjuvant (CFA) inoculated SJL/J mice and from normal mice were cultured for 7 days with medium, with cyclosporin A (CsA), or with CsA and antigen (BP or purified protein derivative of mycobacterium (PPD)). Cultured spleen cells were assayed for suppressor activity in vitro by coculture with BP-specific and PPD-specific helper T cell lines derived from SJL/J mice. Immunized donor spleen cells cultured with cyclosporin A (CsA) and BP were potent inhibitors of T cell line proliferation, and suppressor activity was increased 17-fold compared with control splenocytes. The number of suppressor cells required to suppress PPD-specific line proliferation by 50% (I50) was significantly higher than the number required to suppress BP-specific line proliferation, suggesting an antigen-specific component to the suppression. The major effector cell required for suppression was a large granular Mac-1+ cell with the functional characteristics of a macrophage. Suppressor activity persisted after depletion of Thy 1.2+ cells, but suppression was no longer antigen-specific, suggesting that culture of spleen cells with CsA and BP may generate suppressor macrophages which are antigen-nonspecific and Thy 1.2+ suppressor cells which are antigen-specific. These suppressor cells may be important in the regulation of CR-EAE and the techniques described for their generation may prove useful for treatment and prevention of disease.     

Induction of candidicidal activity in mice by immunization with Candida albicans ribosomes

Abstract Mice immunized with ribosomes from Candida albicans are protected against experimental systemic candidiasis. In this study we investigated the candidacidal activity of spleen cells from immunized animals as measured by ⁵¹Cr release from pre-labelled yeast cells. It was found that the anti-candidal cytotoxic activity of splenocytes from immunized mice was significantly higher than that of spleen cells from non-immunized controls with various effector to target (E:T) ratios, but optimal results were obtained with an E:T ratio of 10:1. The cytotoxic activity of splenocytes as measured by the ⁵¹Cr release assay correlated well with the capacity of the cells to inhibit candidal growth as determined by quantitative plating. This candidacidal activity was not antibody dependent but increased killing was obtained by adding fresh (but not heat inactivated) mouse serum. The enhanced candidicidal activity was inhibited by removal of plastic- or nylon-adherent cells from the cell suspension but not by treatment with anti-Thy 1.2 serum and complement. The data indicate that a candidacidal cell population is induced in the spleens of animals immunized with C. albicans ribosomes.     

The heat shock proteins,Hsp70 and Hsp83, of Leishmania infantum are mitogens for mouse B cells

Extending earlier studies, this report demonstrates that Leishmania infantum heat shock proteins (Hsps), Hsp70 and Hsp83, expressed as recombinant proteins fused to the Escherichia coil maltose-binding protein (MBP), are potent mitogens for murine splenocytes. The response was not due to lipopolysaccharide (LPS) because the stimulatory activity of Hsp preparations was sensitive to boiling and trypsin treatments, whereas the corresponding activity of LPS was resistant to both treatments. It was found that in vitro incubation of spleen cells with the Leishmania Hsps leads to the expansion of CD220-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. In fact, splenocytes from B cell-deficient mice did not proliferate in response to the Leishmania Hsps. In contrast, spleen cells from athymic nude mice were significantly stimulated by these recombinant proteins as an indication that the MBP-Hsp70 and MBP-Hsp83 recombinant proteins behave as T cell-independent mitogens of B cells. Furthermore, both proteins were able to induce proliferation on B cell populations purified from BALB/c spleen.     

Natural cytotoxicity against mouse hepatitis virus-infected target cells. I. Correlation of cytotoxicity with virus binding to leukocytes

Spleen cells from uninfected control mice selectively lysed BALB/c 3T3 fibroblasts infected with mouse hepatitis virus (MHV), a murine coronavirus. Lysis of infected cells occurred within 3 hr, and histocompatibility between effector and target cells was not required. This natural, cell-mediated, virus-associated cytotoxicity differed from NK cell- and T cell-mediated lysis. Spleen cells from animals infected with MHV were enriched in NK activity and were more cytotoxic to YAC-1 target cells, but did not show enhanced cytotoxicity for MHV-infected target cells. Spleen cells from beige mice, which are deficient in NK cell activity, were able to lyse MHV-infected target cells, as were spleen cells from nude mice, which are deficient in T cell activity. Lysis of MHV-infected target cells could be mediated by cells from the spleen and, to a lesser extent, by cells from the bone marrow, but not by resident peritoneal cells or thymocytes. We suggest the term "virus killer (VK) activity" for this phenomenon. VK activity of splenocytes from different mouse strains correlated with the ability of the splenocytes to bind purified radiolabeled MHV virions. MHV virions caused agglutination of spleen leukocytes from susceptible mouse strains, indicating that leukocyte agglutination or adsorption may provide a useful assay for coronaviruses such as MHV which lack hemagglutinating activity. SJL mouse splenocytes did not bind MHV and did not lyse infected targets. MHV bound relatively well to splenocytes of other mouse strains, but poorly to thymocytes and erythrocytes. Binding of MHV to leukocytes was not influenced by 6 mM EDTA or EGTA, indicating a lack of requirement for Mg++ or Ca++. VK activity was also resistant to EDTA and EGTA, in contrast to NK activity, which was sensitive to those chelating agents. VK activity was also unaffected by actinomycin D, cycloheximide, or puromycin, indicating that new protein synthesis was not required for lysis. Antibody to interferon-alpha/beta did not block lysis, nor was there substantially enhanced lysis mediated by leukocytes from mice infected with virus and thus exposed to high levels of interferon. VK activity was blocked by antibody directed against the peplomeric glycoprotein E2 of MHV. VK activity required infected target cells, because cells with adsorbed MHV virions were not lysed by splenocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Release of immature cells from the thymus during solid tumor growth: identification by assay of TdT activity.

In vivo anti-tumor activity of spleen cells from C3H/eb mice bearing a syngeneic fibrosarcoma was shown previously to decline to an undetectable level and be replaced by tumor-enhancing activity as tumor growth proceeds. In the light of our findings that thymocytes in the early stages of thymic processing can bring about tumor enhancement, we postulated that premature release of thymocytes and their accumulation in the spleen might account for the loss of the anti-tumor response. In the present experiments an injection of thymocytes did in fact cancel the anti-tumor response of reactive splenocytes from tumor-bearing mice. In order to determine whether premature thymocyte release occurs naturally in the tumor-bearing animals, we assayed activity of the enzyme TdT (as a marker for thymus cells) in the spleens of these mice during progressive tumor growth. Cells with TdT activity were clearly evident in the spleens of the tumor-bearing animals, were derived from the thymus, and accumulated in parallel to the loss of anti-tumour reactivity.     

Protective effects of glutathione against lipid peroxidation in chronically iron-loaded mice

To elucidate the protective effects of glutathione against iron-induced peroxidative injury, changes in the hepatic glutathione metabolism were studied in chronically iron-loaded mice. When the diets of the mice were supplemented with carbonyl iron, iron deposition occurred primarily in the parenchymal cells of the liver. In addition, expiratory ethane production was elevated, suggesting an enhancement in lipid peroxidation. In iron-loaded mice, the total hepatic glutathione contents were higher (6.21 +/- 0.53 mumol/g wet wt.) than in control mice (4.61 +/- 0.31 mumol/g wet wt.), primarily due to an increase in the reduced glutathione contents. The value of oxidized glutathione was also higher (98.5 +/- 8.1 nmol/g wet wt.) than in the controls (60.8 +/- 9.5 nmol/g wet wt.), and the ratio of oxidized glutathione to total glutathione increased. The excretion rate of glutathione from the hepatocytes in iron-loaded mice also increased. These observations suggest that chronic iron-loading of mice stimulates lipid peroxidation and oxidation of glutathione and that peroxidized molecules may be catabolized using reduced glutathione.     

Induction of arginase activity in the splenocytes of C3HA mice undergoing partial hepatectomy

In splenocytes of C3HA mice after partial hepatectomy the increase in arginase activity is found. It correlates with the increase in cytotoxic splenocyte activity towards tumor cell-target. The suspension without cells capable of adhesion and phagocytosis shows decrease in arginase activity up to 50%. But the enzyme activity is still higher than in splenocytes of intact mice.     

Glutathione status of rat thymocytes and splenocytes during the early events of their ConA proliferative responses

Glutathione plays an important role in the lymphocyte mitogenic response. We have demonstrated that 2-ME increases the ConA proliferative response of rat splenocytes and in parallel, causes an enhancement of glutathione synthesis in these cells. On the other hand, 2-ME had the same action on the glutathione level of thymocytes during the late phase of their mitogenic response, but it had no effect on the [3H]thymidine uptake of these cells. To clarify this discrepancy and the role of glutathione during the mitogenic response, we studied the glutathione status of thymus cells during the early phase of the ConA-induced proliferative response in the presence or the absence of 2-ME in parallel with that of whole spleen cells and the T cell fraction of splenocytes. During the early events of the mitogenic response, i.e., during the 24th h, we observed a normal 2 GSSG/GSH + 2 GSSG ratio in cultured cells, indicating a normal redox state, and that ConA involved an increased glutathione level in thymocytes but not in whole splenocytes and in splenic T cells. 2-ME had no effect on the glutathione level of stimulated thymocytes during the early phase of the mitogenic response. This phenomenon could be related to an absence of its effect on [3H]thymidine uptake. On the other hand, 2-ME induced an enhancement of the glutathione level and [3H]thymidine uptake in the two types of stimulated splenocytes. This study suggest that thymocytes do not have the same mechanism of glutathione synthesis induction as that which occurs in splenocytes during the ConA proliferative response. This mechanism could be related to the maturation state of the T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Splenocytes Seed Bone Marrow of Myeloablated Mice: Implication for Atherosclerosis

Extramedullary hematopoiesis has been shown to contribute to the pathogenesis of a variety of diseases including cardiovascular diseases. In this process, the spleen is seeded with mobilized bone marrow cells that augment its hematopoietic ability. It is unclear whether these immigrant cells that are produced/reprogrammed in spleen are similar or different from those found in the bone marrow. To begin to understand this, we investigated the relative potency of adult splenocytes per se to repopulate bone marrow of lethally-irradiated mice and its functional consequences in atherosclerosis. The splenocytes were harvested from GFP donor mice and transplanted into myeloablated wild type recipient mice without the inclusion of any bone marrow helper cells. We found that adult splenocytes repopulated bone marrow of myeloablated mice and the transplanted cells differentiated into a full repertoire of myeloid cell lineages. The level of monocytes/macrophages in the bone marrow of recipient mice was dependent on the cell origin, i.e., the donor splenocytes gave rise to significantly more monocytes/macrophages than the donor bone marrow cells. This occurred despite a significantly lower number of hematopoietic stem cells being present in the donor splenocytes when compared with donor bone marrow cells. Atherosclerosis studies revealed that donor splenocytes displayed a similar level of atherogenic and atheroprotective activities to those of donor bone marrow cells. Cell culture studies showed that the phenotype of macrophages derived from spleen is different from those of bone marrow. Together, these results demonstrate that splenocytes can seed bone marrow of myeloablated mice and modulate atherosclerosis. In addition, our study shows the potential of splenocytes for therapeutic interventions in inflammatory disease.     

Lyt-2+ T cell-mediated protection against listeriosis. Protection correlates with phagocyte depletion but not with IFN-gamma production

The transfer of listeria-immune splenocytes into non-immune mice markedly increases host resistance to listeriosis. To study the mechanism for this enhancement, we compared the inflammatory response to infection in nonimmune and adoptively immunized mice. Despite much better containment of bacterial growth, adoptively immunized animals accumulated significantly fewer phagocytes (neutrophils and macrophages) in the spleen and liver than controls. Immune T cells not only inhibited phagocyte accumulation but also reduced the in vitro anti-listerial activity of splenocytes. Significant differences in phagocyte accumulation were observed even when the initial listeria dose was adjusted to produce comparable spleen listeria loads in immune and non-immune animals. However, bone marrow and peripheral blood phagocyte counts were similar in both groups. Depletion of Lyt-2+ cells (using mAb and C) from donor splenocytes prevented the transfer of protection and increased phagocyte accumulation without altering listeria-dependent IFN-gamma production by donor or recipient splenocytes in vitro. L3T4 depletion did not affect host resistance or phagocyte accumulation even though it reduced in vitro interferon production by donor cells. Hence the different effects of L3T4+ and Lyt-2+ cells in vivo cannot be explained simply by variations in IFN production. We suggest this paradoxical suppression of phagocyte accumulation during adoptive transfer may reflect lysis of bacteria-laden phagocytes by listeria-specific Lyt-2+ cells in vivo. Selective destruction of older, heavily infected cells might contribute to host resistance by eliminating a potential site for intracellular proliferation of bacteria.     

Pro-carcinogenic activity of beta-carotene, a putative systemic photoprotectant.

Beta-carotene is a strong singlet oxygen quencher and antioxidant. Epidemiologic studies have implied that an above average intake of the carotenoid might reduce cancer risks. Earlier studies found that the carotenoid, when added to commercial closed-formula rodent diets, provided significant photoprotection against UV-carcinogenesis in mice. Clinical intervention trials found that beta-carotene supplementation evoked no change in incidence of nonmelanoma skin cancer. However, when smokers were supplemented with the carotenoid a significant increase in lung cancer resulted. Recently, employing a beta-carotene supplemented semi-defined diet, not only was no photoprotective effect found, but significant exacerbation of UV-carcinogenesis occurred. Earlier, a mechanism, based upon redox potential of interacting antioxidants, was proposed in which beta-carotene participated with vitamins E and C to efficiently repair oxy radicals and, thus, thought to provide photoprotection. In this schema, alpha-tocopherol would first intercept an oxy radical. In terminating the radical-propagating reaction, the tocopherol radical cation is formed which, in turn, is repaired by beta-carotene to form the carotenoid radical cation. This radical is repaired by ascorbic acid (vitamin C). As the carotenoid radical cation is a strongly oxidizing radical, unrepaired it could contribute to the exacerbating effect on UV-carcinogenesis. Thus, vitamin C levels could influence the levels of the pro-oxidant carotenoid radical cation. However, when hairless mice were fed beta-carotene supplemented semi-defined diet with varying levels of vitamin C (0-5590 mg kg(-1) diet) no effect on UV-carcinogenesis was observed. Lowering alpha-tocopherol levels did result in further increase of beta-carotene exacerbation, suggesting beta-carotene and alpha-tocopherol interaction. It was concluded that the non-injurious or protective effect of beta-carotene found in the closed-formula rations might depend on interaction with other dietary factors that are absent in the semi-defined diet. At present, beta-carotene use as a dietary supplement for photoprotection should be approached cautiously.