Modulation of B and T cell subsets in mice treated with fractionated total lymphoid irradiation. III. Spleen and thymus dependency of B cell maturation processes

The role of spleen and thymus in maturation processes of B cell subpopulations was examined using mice which underwent fractionated total lymphoid irradiation (TLI). (BALB/BL)F₁ mice received 200 rds lymphoid irradiation daily for 8 days. Mice were spleen-shielded or -unshielded, or thymus-shielded or -unshielded during the irradiation. One day after termination of the irradiation, TLI-treated unshielded mice were reconstituted with 10 × 10⁶ spleen, thymus or bone marrow cells of normal untreated syngeneic mice. Two and a half months after termination of the treatment, the ability of the treated mice to produce in vivo anti-trinitrophenyl (TNP)-Ficoll antibodies, and the capability of spleen cells of those mice to respond in vitro to dextran sulfate and lipopolysaccharide was checked. In parallel, stained spleen cells were analyzed on the fluorescence-activated cell sorter. The results indicate that B cell maturation occurs only in mice where the spleens were shielded during TLI-treatment or in TLI-treated mice reconstituted with spleen cells of normal untreated mice. In these mice, the light scatter and the fluorescence distribution profiles were the same as those obtained from spleen cells of control mice: the treated mice gave a high anti-TNP-Ficoll antibody response and the proliferative response of the cells was low to dextran sulfate and high to lipopolysaccharide. Thymus shielding during TLI treatment or reconstitution of TLI-treated mice with thymus or bone marrow cells could not abolish the blockage of B cell maturation processes. These findings indicate that the spleen plays an obligatory role in B cell subset maturation pathways, whereas the thymus appears to play no essential role in these processes.     

Reduced volume-regulated outwardly rectifying anion channel activity in ventricular myocyte of type 1 diabetic mice

The currents through the volume-regulated outwardly rectifying anion channel (VRAC) were measured in single ventricular myocytes obtained from streptozotocin (STZ)-induced diabetic mice, using whole-cell voltage-clamp method. In myocytes from STZ-diabetic mice, the density of VRAC current induced by hypotonic perfusion was markedly reduced, compared with that in the cells form normal control mice. Video-image analysis showed that the regulatory volume decrease (RVD), which was seen in normal cells after osmotic swelling, was almost lost in myocytes from STZ-diabetic mice. Some mice were pretreated with 3-O-methylglucose before STZ injection, to prevent the STZ’s β cell toxicity. In the myocytes obtained from such mice, the magnitude of VRAC current and the degree of RVD seen during hypotonic challenge were almost normal. Incubation of the myocytes from STZ-diabetic mice with insulin reversed the attenuation of VRAC current. These findings suggested that the STZ-induced chronic insulin-deficiency was an important causal factor for the attenuation of VRAC current. Intracellular loading of the STZ-diabetic myocytes with phosphatidylinositol 3,4,5-trisphosphate (PIP3), but not phosphatidylinositol 4,5-bisphosphate (PIP2), also reversed the attenuation of VRAC current. Furthermore, treatment of the normal cells with wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, suppressed the development of VRAC current. We postulate that an impairment PI3K-PIP3 pathway, which may be insulin-dependent, is responsible for the attenuation of VRAC currents in STZ-diabetic myocytes.     

Bone marrow origin of mucosal mast cells

Infection with the intestinal parasite Nippostrongylus brasiliensis stimulates an accumulation of mucosal mast cells (MMC) in the villi of the small intestine of normal but not athymic or W/Wv anemic mice. W/Wv mice are congenitally deficient in both MMC and skin and connective tissue mast cells (CTMC). Athymic mice have normal or elevated numbers of CTMC but are severely deficient in MMC. CTMC derive from the bone marrow. To determine the origin of MMC, athymic and W/Wv mice were given various hematopoietic or lymphoid tissues from normal littermate or beige mice and the MMC response to N. brasiliensis infection was evaluated. The MMC defect in athymic mice was repaired by grafts of thymus cells, thymus gland, or spleen cells, but not by bone marrow cells or anti-Thy 1-treated bone marrow or spleen cells. The MMC and CTMC defects of W/Wv mice were repaired by grafts of bone marrow, spleen cells, or anti-Thy 1-treated bone marrow or spleen cells. Neither the MMC nor the CTMC defect in W/Wv mice was repaired by grafts of thymus cells or thymus glands. These results indicate the following, MMC, like CTMC, derive from the bone marrow and not from the thymus. MMC require a thymic influence for development. Athymic mice possess bone marrow precursors for both MMC and CTMC but lack a thymus-dependent component necessary for MMC development. W/Wv mice lack both MMC and CTMC mast cell precursors but possess the thymus-dependent component required for MMC development.     

Effect of cortisone and X-irradiation on cellular depletion and regeneration in the thymus of mice: experimental discrimination between thymus lymphocyte precursors in the bone marrow and in the thymus.

The effect of cortisone treatment on the ability of bone marrow cells to repopulate X-irradiated thymus was investigated. In one experimental series, groups of mice were treated first with cortisone and then irradiated with or without bone marrow protection. Mice treated with either cortisone or radiation alone served as controls. During an initial, bone marrow independent phase of thymus regeneration, cortisone had a stronger inhibitory effect on the cellular regeneration of the organ than irradiation. On the other hand, during a subsequent bone marrow dependent phase, thymus regeneration was impaired by radiation exposure but not by cortisone treatment. In another experimental series, irradiated mice were transplanted with bone marrow cells in different numbers from syngeneic donors which had either been treated with cortisone or were left untreated. Twenty days later the cell number was consistently larger in the thymus of animals which has been transplanted with cortisone treated bone marrow than in the animals transplanted with untreated bone marrow. It is concluded that the thymus lymphocyte precursors in the bone marrow and the early precursors of thymocytes in the thymus differ with regard to their sensitivity to cortisone and radiation and, therefore, may represent two distinct cell types.     

Age dependent reaction of mouse thymus cells with autologous and syngeneic erythrocytes.

The age dependent events influencing the rosette forming capacity of Balb/c thymus and spleen cells against autologous or syngeneic erythrocytes were examined. A large number of autologous and syngeneic rosette forming cells (RFC) were observed in normal Balb/c mice in vitro. RFC were significantly greater in the thymus than in the spleen. The rosette forming T-cells (T-RFC) have the following characteristics: newborn Balb/c thymus has T-cells which react syngeneic erythrocytes from older donors. The T-RFC showed broad cross-reactivity with erythrocytes from other mouse strains but low reactivity with human or sheep erythrocytes. The auto and syngeneic T-RFC could be enhanced by non-specific serum proteins (FCS or BSA) or EDTA but was effectively inhibited by normal mouse serum. T-RFC resided in the cortisone sensitive population. The data indicate that the development of autologous and syngeneic rosette formation of thymus cells is dependent on the age of the erythrocyte donor. The age dependent change on the erythrocyte surface occurred relatively early in the life of the animal. The results also imply that certain subpopulations of thymus lymphocytes are capable of recognizing possible surface antigenic changes of the erythrocytes.     

Effect of cyclophosphamide on the immune response to Pseudomonas aeruginosa in mice.

Natural resistance in mice to Pseudomonas aeruginosa was decreased 10-fold with a single dose of 300 mg of cyclophosphamide (CY) per kg intraperitoneally. Mice were resistant to infection when immunized actively with Pseudomonas vaccine or passively with Pseudomonas immune serum before receiving CY. Syngeneic spleen, thymus and/or bone marrow cells were transfused into CY-treated recipient mice. Protective anti-Pseudomonas antibody was elicited in the recipient mice when they were vaccinated 1 day after receiving normal spleen cells and challenged 8 days after vaccination. When 1.6 X 10(7) normal thymus and bone marrow cells were infused before vaccination, 69% of the recipients of both cell preparations responded serologically compared with 15 and 27% of those receiving either thymus or bone marrow cells, respectively. CY-treated thymus or bone marrow cell recipients were resistant to Pseudomonas infection when 6 X 10(7) of either cell population was transfused.     

Antigenic differences between spleen-seeking and lymph node-seeking thymus cells

Thymus cells of mice of the C3H and A strains were labeled with ⁴¹Cr and exposed in vitro to either normal mouse serum or to various antisera and the cells injected intravenously into syngeneic recipients. Exposure of thymus cells to various sera was found to have different effects on the cell subpopulations migrating to either the lymph nodes or the spleen. Anti-H-2 serum had a marked inhibitory effect on the migration of thymus cells to lymph nodes, and a much weaker effect on their migration to the spleen. Anti-TH serum had a slightly stronger inhibitory effect on spleen-seeking, as compared to lymph node-seeking thymus cells. Anti-Ly serum had a strong inhibitory effect on both migration streams. Anti-TL serum had a stronger inhibitory effect on spleen-seeking thymus cells than on lymph node-seeking cells, a difference which became even more pronounced in cells exposed to Anti-TL serum and complement. Normal guinea pig serum had a marked inhibitory effect on thymus cells migrating to the spleen, but hardly affected the lymph node-seeking thymus cells. These results indicate that the thymus contains two major subpopulations of cells of differing antigenicity, which migrate to either the spleen or the lymph nodes. It is suggested that the different properties of T cells found in the spleen and in the lymph nodes result from the migration of different subpopulations of thymus cells to these organs.     

Hapten-specific tolerance in mice. II. Adoptive transfer studies and evidence for unresponsiveness in the B cells

The suppression fo the anti-NIP ((4-hydroxy-5-iodo-3-nitrophenyl)acetyl) plaque-forming cell (PFC) response elicited in mice by treatment with NIP-coated syngeneic erythrocytes could be transferred by spleen cells into irradiated recipients. This was evidenced by the lack of an indirect anti-NIP PFC response 7 days after cell transfer and challenge with NIP. FGG (fowl IgG). This state of specific unresponsiveness could be serially transferred by spleen cells into a second irradiated recipient. The hapten-specific suppression in the first recipient could be reversed by addition of normal spleen cells, but not by cortisone-resistant thymus cells. The lesion appears to be in the T cell-depleted population, since the suppression could be reversed by supplementing with T cell-depleted normal spleen cells. In vitro incubation of spleen cells from tolerant animals did not restore the capacity of these cells to produce an anti-NIP PFC response in irradiated recipients. In vitro incubation of normal spleen cells with sera from tolerant animals did not prevent these spleen cells from producing a normal anti-NIP PFC response in irradiated recipients. The adoptive secondary response response to NIP. FGG was inhibited by injection of NIP-coated syngeneic erythrocytes on the same day as the adoptive transfer of the NIP. FGG primed spleen cells and the challenging antigen. It would seem that NIP-coupled syngeneic erythrocytes, which are presumably poor stimulators of T cells, can suppress NIP-specific B cells, perhaps by gaining direct access to the surface of these cells.     

Studies on the adsorbability of graft-versus-host-reactive lymphocytes

A certain proportion of normal lymphoid cells from organs of different anatomical origin can react against allogeneic histocompatibility antigens, e.g. they can mediate a graft-versus-host (GVH) reaction. The present study was undertaken in order to obtain information about the antigen-specific receptors on GVH-reactive lymphocytes. We used fibroblast monolayers as cellular immunoadsorbents.

The results showed that with the methods employed we could not adsorb specifically GVH-reactive lymphocytes in a reproducible way, irrespective of whether these cells were from normal lymphoid organs like spleen, lymph nodes, thymus and bone marrow, from artificial sources like thymus and spleen of anti-thymocyte antiserum treated mice or from spleens of semi-allogeneic, irradiated, thymocyte-reconstituted mice. The most pronounced effect of adsorption on fibroblast monolayers on normal lymphocytes was a non-specific enhancement of their GVH potential.

These findings are discussed on the basis of recent results showing both syner-gistic and antagonistic effects of T-lymphocyte subpopulations.

     

Hapten-specific tolerance: unresponsiveness in the T cell-depleted population

Complete and specific suppression of the anti-NIP [(4-hydroxy-5-iodo-3-nitro-phenyl)acetyl] indirect plaque-forming cell response to NIP-fowl IgG was obtained in mice by pretreatment with NIP-coated syngeneic erythrocytes. The complete suppression occurred within 5 to 7 days and was evident after adoptive transfer of spleen cells into irradiated mice. The state of unresponsiveness was reversed, not by thymus cells, but by supplementing with T cell-depleted normal spleen cells. This suggests that the unresponsiveness occurred at the level of the B lymphocyte population.     

Lymphocyte activation: I. Response of T and B lymphocytes to phytomitogens

The selectivity of phytomitogens for T (thymus derived/dependent) and B (`bursa-equivalent' dependent/derived) lymphocytes from the mouse spleen has been investigated. Responses of normal spleen cell cultures were compared with those of cultures of selected B cells. The latter were obtained from three sources (1) spleen cells of mice that had been thymectomized as adults, lethally irradiated and reconstituted with syngeneic bone marrow cells pretreated with anti-θ serum (2) spleen cells from congenitally athymic (`nude') mice and (3) spleen cells from normal mice treated with anti-θ serum plus guinea-pig complement prior to culture.

Using a variety of different culture conditions it was shown that B cells respond well to pokeweed mitogen, and poorly if at all to phytohaemagglutinin. Responsiveness to the latter mitogen in normal spleen cell cultures appears to be a property of T cells.

     

Comparative study on the anticellular and antiviral effects of interferon and the haemopoietic stem cell inhibition factor.

Pretreatment with crude interferon preparations obtained from suspension cultures of bone marrow, spleen and thymus cells or from mouse L-cell cultures or with mouse serum interferon preparations did not change the colony-forming activity of bone marrow cells on syngeneic transplantation to lethally irradiated mice. Preparations of L-cell culture interferon, dialysed and purified by carboxymethyl-Sephadex (G-25) column chromatography, showed an inhibitory effect on exogenous colony formation by bone marrow cells. The results suggested the presence in crude interferon preparations of a substance either inhibiting the anticellular effect of interferon or stimulating colony formation. The factor produced by thymus cells following their treatment with antilymphocyte serum inhibited colony formation by bone marrow cells and, unlike interferon, possessed no antiviral activity when tested in cell cultures.     

DIFFERENTIATION OF MOUSE T-LYMPHOCYTES 1. CHARACTERIZATION OF THYMOCYTE PRECURSORS IN BONE MARROW BY ONE G VELOCITY SEDIMENTATION

Bone marrow cells were transferred into 700 RX-irradiated syngeneic mice, and reganeration of the thymus was demonstrated by determining the total amount of DNA in the thymus. The amount of DNA in the regenerated thymus 9 days after X-irradiation paralleled the number of cells injected, the range being 0 to 10⁶ cells. These results indicate that regeneration during this period reflects the proliferation of thymocyte precursors existing in bone marrow cells, therefore such can be used as an indicator to determine the number of thymocyte precursors.
Thymocyte precursors were separated from spleen colony forming cells and soft agar colony forming cells by one G velocity sedimentation. Thymocyte precursors (more than 4.3 mm/hour sedimentation rate) sedimented faster than did spleen colony forming cells (peak at about 3.0 mm/hour) and Thy-1 antieen was already evidenced on the cell surface as demonstrated by complement dependent cytotoxicity. Soft agar colony forming cells also sedimented faster, suggesting that cells destined to become one type of cell are larger than multi-potential stem cells.     

Stress-induced alterations in the programmed natural cycles of post-natal lymphoid organ development in C57BL/6 mice: Evidence for a regulatory feedback relationship between bone marrow and thymus

This study investigated some effects of weaning and immobilization stress in C57BL/6 mice aged 22-68 days, i.e., over a period including activation of the hypothalamus-pituitary-adrenal (HPA) axis and puberty. Specifically, the study evaluated the evolution, over the referred age interval, of a set of variables (body, thymus, spleen and axillary lymph nodes weights, the proportion of lymphoid cells in the bone marrow, the relative chemoattraction capacity of thymic supernatants for lymphoid cells and the migratory capacity of bone marrow lymphoid cells) in either weaned mice or weaned mice subjected to immobilization stress, compared to "non-stressed" unweaned mice. Cyclic patterns, observed for most variables in unweaned mice, were especially pronounced in two cases: the relative migratory capacity of bone marrow lymphoid cells collected at different ages towards neonatal thymic supernatant, and the relative chemoattraction capacity of thymic supernatants of different ages as tested against a sample of bone marrow lymphoid cells from mice aged 35 days. Weaning stress tended to intensify the involution stages of the cycles in thymus, spleen and lymph node weight, but increased the relative proportion of lymphoid cells in the bone marrow cell population. Both types of exogenous stress tended to affect cycle phase, i.e., cycle peaks and troughs were shifted in time. Correlations were observed between patterns seen in the thymus and bone marrow, suggesting the existence of an autoregulatory feedback loop governing pre-T cell migration and bone marrow/thymus homeostasis. These results also suggest that exogenous stress acts as a non-programmed regulator, modulating the naturally programmed cyclic patterns.     

Differentiation of mouse T-lymphocytes. 19 Characterization of thymocyte precursors in bone marrow by one G velocity sedimentation.

Bone marrow cells were transferred into 700 R X-irradiated syngeneic mice, and regeneration of the thymus was demonstrated by determining the total amount of DNA in the thymus. The amount of DNA in the regenerated thymus 9 days after X-irradiation paralleled the number of cells injected, the range being 0 to 10(6) cells. These results indicate that regeneration during this period reflects the proliferation of thymocyte precursors existing in bone marrow cells, therefore such can be used as an indicator to determine the number of thymocyte precursors. Thymocyte precursors were separated from spleen colony forming cells and soft agar colony forming cells by one G velocity sedimentation. Thymocyte precursors (more than 4.3 mm/hour sedimentation rate) sedimented faster than did spleen colony forming cells (peak at about 3.0 mm/hour) and Thy-1 antigen was already evidenced on the cell surface as demonstrated by complement dependent cytotoxicity. Soft agar colony forming cells also sedimented faster, suggesting that cells destined to become one type of cell are larger than multi-potential stem cells.     

Thymus grafting in allogeneic radiation chimaeras

CBA/H mice were lethally irradiated and treated with C57BL/H bone marrow cells. 8–14 days thereafter they were grafted under the kidney capsule with thymus derived either from CBA or from C57BL donors, 4–8 days old. Only 8 per cent of non-grafted animals survived 4 months, but 39 per cent of those grafted with CBA thymus, and 48 per cent of those grafted with C57BL thymus survived the same period. Grafted chimaeras of both groups had significantly more leucocytes in the peripheral blood, and more colony-forming units in the bone marrow, than chimaeras without a graft. A thymus graft syngeneic either with the donor or with the recipient of bone marrow improved the reactivity against bacterial antigens of Salmonella, but did not influence the reactivity against tissue antigens of sheep erythrocytes. It is concluded that immunological functions of allogeneic radiation chimaeras can not be restored entirely by stem cells from the bone marrow, but require normal (or even increased) cellular and humoral influence from non-irradiated thymus tissue as well.     

Precursor cells of fibroblasts detected by in vitro cloning of cells from hematopoietic organs of normal and irradiated mice

Colonies of fibroblasts are formed in monolayer cultures of bone marrow, spleen, and thymus cells of adult mice with an efficiency of colony formation (per 10⁵ cells) of 2.2 for bone marrow, 0.20 for spleen, and 0.16 for thymus. On irradiation of mice with a dose of 150 R, about half of the fibroblasts colony-forming units in the bone marrow die; during the next 6 days their number falls a further fivefold, with a return to the normal level 25 days after irradiation.Laboratory of Immunomorphology, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 5, pp. 614–617, May, 1976.     

Strain differences in the immune response of mice: III. A raised tolerance threshold in NZB thymus cells

Irradiated (NZB×BALB/c)F1 mice were injected with syngeneic bone marrow cells, syngeneic or parental thymus cells, and sheep red cells. The antibody plaque-forming cell response depended on the number of sheep cells and the age and strain of the thymus cells. Young or adult BALB/c thymus, and young hybrid thymus, responded best to low numbers of sheep cells; with higher numbers they became tolerant. Adult hybrid thymus, and young or adult NZB thymus, responded better to high numbers of sheep cells. Hybrid mice irradiated and restored with BALB/c bone marrow developed thymus cells with the reactivity of BALB/c thymus. It is argued that NZB mice, and older hybrids, may develop autoimmunity because of an abnormality of tolerance induction manifested in their thymus cells, but of bone marrow origin.     

Effects of GnRH Antagonist on Lymphocyte Subpopulations in Primary and Secondary Lymphoid Tissues of Female Mice

PROBLEM: GnRH analogs are playing an increasing role in the treatment of many clinical disorders. Recent studies have indicated that GnRH agonists suppress immune function in mice in vivo. The present study investigated the effects of GnRH antagonist on functional lymphocyte subsets of mice in vivo. METHOD: Three- and 10-wk old female mice received 10 μg of Nal-Glu daily for 15 and 30 days; changes in the immunophenotypic expression of lymphocytes from thymus, bone marrow, spleen and blood were analyzed by flow cytometry. RESULTS: The administration of GnRH antagonist to pre- and postpubertal female mice induced slight increases in lymphocyte subpopulations in primary and secondary lymphoid tissues. These effects are opposite those obtained with GnRH agonist in our earlier studies in mice. CONCLUSIONS: Assuming similar effects in humans and rodents, the gonadal steroid suppression achieved by GnRH antagonist treatment has no apparent suppressive effects on the immune system.     

Selectivity in the Effects of Indomethacin on Bcg-Activated Suppressor Cell Populations

Intravenous inoculation of BCG into C57 B1/6 mice activated natural suppressor cells in the bone marrow and induced suppressor cells in the spleen. The suppressor activity of these cell populations was determined by co-cultivating them with normal lymphocytes being immunized against allogeneic P815 cells in vitro. Six million cells from the spleen or bone marrow of BCG treated mice inhibited by more than 50% the alloimmunization of twenty million normal syngeneic lymphocytes. The suppressor cells were found in the nylon wool adherent population in spleen and in both the nylon wool adherent and non-adherent POulations in bone marrow. Indomethacin, at a concentration of 10^--⁶M, completely blocked the suppression generated by unfractionated spleen or adherent spleen from BCG treated mice. However, 10^--⁶M and higher concentratlons of indomethacin only partially blocked the suppression generated by unfractionated marrow or adherent marrow from BCG treated mice. The suppression generated by non-adherent marrow from BCG treated mice was completely insensitive to indomethacin.