Effect of splenectomy on the development of neonatal lymphoid organs transplanted to animals of varying age]

The effect of splenectomy on the development of newborn thymus and spleen grafted under the kidney capsule of young and old mice has been investigated. Preliminary splenectomy is shown to increase cell counts in grafted spleen that is more conspicuous in young recipients as compared with old ones. This result suggests a decrease with age in the inhibitory effect of the host spleen on the maturation of spleen grafted from newborn donor. Combined transplantation of newborn thymus and spleen has revealed a decrease of cell counts in the donor spleen grafted to the young splenectomized recipients and, on the contrary, increase of this parameter in old ones. Immune response in donor spleen with combined transplantation of the thymus to the old splenectomized recipients is much higher as compared with the same parameter in recipient without splenectomy. It is concluded that partial destruction of the old immune system is essential for its correction.     

Effects of the age of the recipient on the outcome of transplantation of lymphoid organs from newborn donors

The comparison of developmental capacities of simultaneously grafted thymus and spleen taken from newborn CBA mice in young and old macroenvironment was attempted using the method of heterotopic chimera construction. The perceptible augmentation of immune response in both host and donor spleens was obtained in case of transplantation into the young animals, and completely opposite effect, i. e. well-marked immunosuppression in case of old recipients. Preliminary removal of host spleen results in abolition of this suppressive effect in old animals. Moreover, immune response in donor spleen following thymus transplantation into the old splenectomized recipients was significantly higher compared to the same one without additional thymus graft. Taken together, these findings indicate that despite the existence of some potency for autonomous development the eventual effect of neonatal lymphoid organs maturation strongly depends upon the age of system in which this maturation does take place.     

Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34⁺ cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation.Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34⁺ cord blood stem cell preparations.     

Cell lines from old immunodeficient donors give normal responses in young recipients.

Two different immune responses were compared in spleen cells obtained from old and young CBA/HT6J mice. Spleen cells from old mice (23 to 33 months) responded about half as well as did spleen cells from young mice (4 to 10 months) in the adoptive transfer anti-sheep red blood cell (SRBC) plague-forming assay, and caused slightly less than half the uptake of tritiated thymidine in response to phytohemagglutinin (PHA) in vitro. Marrow stem cell from some of the old and young mice whose splenic immune responses were tested were transplanted into irradiated young CBA/CaJ recipients. Seven to 17 weeks later these same immune responses were tested in the spleen cells of these young recipients, and the T6 chromosome marker was used to identify donor cells. Old animals' responses varied greatly, perhaps due to suppressing cells or factors in some individuals. Therefore, cells were never pooled and the responses of receipients were compared to the responses of the donor whose marrow had populated them. The response for a particular old donor, or for the recipients of its stem cells, was divided by the response for the young control used with that donor, or for its stem cell recipients. This was called the old/young ratio. With original donors with an old/young ratio for the SRBC response of (mean +/- S.D.) 0.35 +/- 0.14, The old/young ratio for that same response in the recipients was significantly improved to 1.26 +/- 0.71. In original donors with an old/young ratio for the PHA response of 0.44 +/- 0.17, the old/young ratio in the recipients improved significantly to 0.86 +/- 0.27. Thus, little or none of the decline with age in these immune responses was intrinsic to the old lymphoid stem cells.     

Development of the dendritic cell system during mouse ontogeny

Based on the view that the efficacy of the immune system is associated with the maturation state of the immune cells, including dendritic cells (DC), we investigated the development and functional potential of conventional DC and plasmacytoid pre-DC (p-preDC) in spleen, thymus, and lymph nodes during mouse development. Both CD11c+ DC and CD45RA+ p-preDC were detected in small numbers in the thymus as early as embryonic day 17. The ratio of DC to thymocytes reached adult levels by 1 wk, although the normal CD8alpha+ phenotype was not acquired until later. Significant, but low, numbers of DC and p-preDC were present in the spleen of day 1 newborn mice. The full complement of DC and p-preDC was not acquired until 5 wk of age. The composition of DC populations in the spleen of young mice differed significantly from that found in adult mice, with a much higher percentage (50-60% compared with 20-25%) of the CD4-CD8alpha+ DC population and a much lower percentage (10-20% compared with 50-60%) of the CD4+CD8alpha- DC population. Although the p-preDC of young mice showed a capacity to produce IFN-alpha comparable with that of adult mice, the conventional DC of young mice were less efficient than those of their adult counterparts in IL-12p70 and IFN-gamma production and in Ag presentation. These results suggest that the neonatal DC system is not fully developed, and innate immunity is the dominant form of response. The complete DC system required for adaptive immunity in the mouse is not fully developed until 5 wk of age.     

Role of immunity in age-related resistance to paralysis after murine leukemia virus infection.

Resistance to the paralytic effects of a wild mouse (Cas-Br-M) murine leukemia virus infection develops with age and is complete by 10 days of age in susceptible NFS mice. The possibility that cell-mediated immunity plays a significant role in this resistance was suggested by the observation that treatment of 10-day-old mice with antithymocyte serum rendered them susceptible to paralysis. By comparison, mice rendered incapable of generating a humoral immune response by treatment from birth to 1 month of age with anti-immunoglobulin M serum did not develop paralysis after challenge with virus at day 10. Transfer of unseparated and T-cell-enriched populations of Cas-Br-M murine leukemia virus-immune spleen cells protected neonatally infected NFS recipients from paralysis; transfer of Cas-Br-M murine leukemia virus-immune populations enriched for B cells delayed the onset but did not ultimately protect neonatally infected NFS mice from paralysis. Transfer of naive adult spleen cells had no protective effect in neonatally infected NFS mice. High-level virus replication occurred in the spleens and brains of all mice that developed paralysis regardless of treatment; low-level virus replication in spleen and barely detectable replication in brain occurred in mice that remained clinically normal. These studies suggest that the age-acquired resistance to the paralytic effect of Cas-Br-M murine leukemia virus infection is immunologically mediated and that T cells may play a major role.     

Analysis of certain mechanisms of antigen-specific suppression of the immune response

CBA mice were immunized with sheep red blood cells (SRBC) to obtain immune spleen cells (ISc) which were used to suppressor cells. Administration of ISC to intact syngeneic recipients on the immunization day led to a more powerful suppression of the immune response as compared to that seen one day after antigen injection. Four days after immunization the animals' immune response was not liable to be suppressed. ISC extract possessed similar effects with respect to the immune response of normal spleen cells which were transplanted to the cyclophosphamide-treated recipients. The immune response of spleen cells from mice immunized with SRBC in a dose of 10(6) was less liable to be suppressed. Hyperimmune spleen cells from donors immunized with SRC in a dose of 10(9) were insensitive to ISC or to the extract. Experiments with the use of adoptive transfer of a mixture of immune and intact T- and B-cells have disclosed that B-cells from hyperimmune donors were resistant to suppression. Therefore, B-lymphocytes are the most probable target cells exposed to T-suppressors in the given system. The mechanism is discussed of the selective effect of T-suppressors on B-cells in the course of the immune response development during immunization with high doses of antigen.     

Candidate ligand for the c-kit transmembrane kinase receptor: KL, a fibroblast derived growth factor stimulates mast cells and erythroid progenitors.

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor for an unidentified ligand and is allelic with the murine white-spotting locus (W). W mutations affect melanogenesis, gametogenesis and hematopoiesis during development and in adult life. Cellular targets of W mutations in hematopoiesis include distinct cell populations in the erythroid and mast cell lineages as well as stem cells. In the absence of interleukin-3 (IL-3) mast cells derived from normal mice but not from W mutant mice can be maintained by co-culture with 3T3 fibroblasts. Based on the defective proliferative response of W mast cells in the 3T3 fibroblast co-culture system it had been proposed that fibroblasts produce the c-kit ligand. We have used a mast cell proliferation assay to purify a 30 kd protein, designated KL, from conditioned medium of Balb/3T3 fibroblasts to apparent homogeneity. KL stimulates the proliferation of normal bone marrow derived mast cells but not mast cells from W mice, although both normal and mutant mast cells respond similarly to IL-3. Connective tissue-type mast cells derived from the peritoneal cavity of normal mice were found to express a high level of c-kit protein on their surface and to proliferate in response to KL. The effect of KL on erythroid progenitor cells was investigated as well. In combination with erythropoietin, KL was found to stimulate early erythroid progenitors (BFU-E) from fetal liver and spleen cells but not from bone marrow cells of adult mice and from fetal liver cells of W/W mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of lithium chloride treatment on cell-mediated immunity in mice.

The immunomodulatory effect of lithium chloride (LiCl) administered i.p. to adult mice (150 mg/kg/day) on cellular immunity in vivo was investigated. A short (6-day) treatment with LiCl of either spleen cell donors in semiallogeneic or xenogeneic GVH reaction, or recipients in semiallogeneic HVG reaction, significantly diminished the early sings of local cell-mediated immunity. A short-term LiCl treatment of donor mice also abrogated systemic GVH reaction in newborn F1 recipients, while a treatment prolonged for 20 days did not alter the high GVH response of spleen cells typical for untreated donors. Thus, a striking time-dependent efficacy of LiCl on the reduction of GVH reaction was found.     

Suppressive effect of the blast cells of AKR strain mice on antibody formation in vivo and lymphocyte proliferation in vitro

Blast cells obtained from the "erythropoietic spleen" of FG-stimulated young mice and cells accumulating in the spleens of preleukemic AKR mice have a marked suppressive effect on spontaneous and mitogen-induced proliferation of young mouse splenocytes in vitro and suppress the development of humoral immune response in immunized recipients during syngeneic transfer in vivo. Some disturbances in erythron system in preleukemic AKR mice manifested in the accumulation of immature erythroid precursors which are suppressors of immunocompetent lymphocytes are suggested to be a pathogenetic link in the development of leukemia.     

Effect of syngenic lymphocytes on allogenic inhibition of hematopoietic stem cells of embryonic liver]

The transplantation of liver from the embryos and newborn C57BL-6 mice to the lethally irradiated hybrids (CBA X C57BL/6) F1resulted in 90% allogenic inhibition of the colony-forming activity of the donor elements. The degree of allogenic inhibition of liver cells of 19 days old embryos and newborn mice may be changed with the help of syngenic lymphocytes of adult mice or delayed transplantation of cells 72 hrs following the irradiation of recipients but these procedures proved to be ineffective with the liver cells of 13 and 16 days old embryos. A suggestion is put forward to the effect that the allogenic inhibition is based on the active reaction of recipient hybrids (CBAXXC57BL/6) F1 to the stem hemopoietic cells of C57BL/6 mice.     

Nature of cells forming erythroid colonies in agar after stimulation by spleen conditioned medium

Erythroid colony formation in agar cultures of CBA cells was stimulated by the addition of pokeweed mitogen-stimulated C57BL spleen conditioned medium. Both 48-hour colonies ("48-hour benzidine-positive aggregates") and day 7 large burst or unicentric erythroid colonies ("erythroid colonies") developed, together with many neutrophil and/or macrophage colonies. In CBA mice, the cells forming erythroid colonies occurred with maximum frequency (650/10(5) cells) in 10- to 11-day-old yolk sac and fetal liver but were present also in fetal blood, spleen and bone marrow. The frequency of these cells fell sharply with increasing age and only occasional cells (2/10(5) cells) were present in adult marrow. A marked strain variation was noted, CBA mice having the highest levels of erythroid colony-forming cells. The erythroid colony-forming cells in 12-day CBA fetal liver were radiosensitive (DO 110-125 rads), mainly in cycle and were non-adherent, light density, cells sedimenting with a peak velocity of 6-9 mm/hr. These properties are similar to those of other hemopoietic progenitor cells in fetal tissues. The relationship of these apparently erythropoietin-independent erythroid colony-forming cells to those forming similar colonies after stimulation by erythropoietin remains to be determined.     

Regulation of antigen induced changes in cyclic nucleotide levels in NZB/wf1 mice.

Normal C57BL/6J mice respond to the iv injection of antigen with an increase in splenic cAMP at 2 min. NZB/WF₁ mice are predisposed to autoimmune and immunological disorders upon aging. The ability of NZB/WF₁ mice to respond to antigen with an increase in their splenic cAMP level was found to diminish with age. This loss of responsiveness is antigen specific and not due to a loss of adenylate cyclase activity in spleen cells of old NZB/WF₁ mice. The adoptive transfer of spleen cells from unresponsive old mice into responder young mice inhibited the cAMP response to antigen by the recipients. Spleen cells from young responsive mice, on transfer into old nonresponsive NZB/WF₁ recipients, resulted in restoration of the cAMP response to antigen. In both cases, the activity of donor cells was dependent on the transfer of T cells. These results indicate that populations of T cells participate in the regulation of the cAMP response to antigen by NZB/WF₁ mice. The response of old mice could also be restored by treatment with indomethacin, and also the spleen cells of such treated donors failed to suppress the cAMP response of young recipients. Together, the results suggest a role for prostaglandins in regulating the cAMP response to antigen.     

Characterization of the Growth Factor Activity of Amniotic Fluid on Cells from Hematopoietic and Lymphoid Organs of Different Life Stages

We investigated the proliferation-promoting effects of murine amniotic fluid (MAF) on in vitro cultured cells originally obtained from murine hematopoietic and lymphoid organs at different life stages. MAF promoted proliferation of the fetal liver cells (FLC), newborn spleen cells and adult bone marrow cells. The proliferation-promoting activity of MAF was extended to liver cells and spleen cells from mice younger than 2 weeks old. MAF did not, however, promote the proliferation of newborn or adult thymocytes, or of spleen cells, liver cells or peritoneal cells from 2-week-old or older mice. Rather, it partially inhibited the proliferation of spleen cells, thymocytes and peritoneal cells from 1-year-old mice. These results suggest that MAF contains growth factors for hematopoietic stem cells but not for either mature or immature T lymphocytes. Supporting this view, the MAF activity was partially neutralized by a polyclonal anti-mouse stem cell factor (SCF) antibody. Moreover, the immunoblotting of MAF against anti-mouse SCF antibody revealed a band at 30–32 kDa corresponding to the previously reported SCF. Interestingly, MAF was able to maintain FLC and adult bone marrow cells alive in culture for a relatively long time (2 weeks). The MAF activity was further shown to be partially and cell type-dependently antagonized by TNF-α and TGF-β. These results provided evidence that MAF contains potentially multiple growth factors preferentially affecting the early stage of hematopoiesis, one of which is SCF.     

Ontogenic development of the reactivity of macrophages to antigenic stimulation

Experiments were performed to test the ability of macrophages from newborn mice to participate in immune reactions. It was found that peritoneal cells from 4-day-old mice injected at birth with thioglycollate did not reconstitute reactivity to Shigella in adult, irradiated mice, while normal adult macrophages did.The total yield of peritoneal exudate cells (PEC) was relatively low, yet the number of precursor cells of macrophages in the newborn spleen was found in significantly higher concentrations than in the adult. The number of precursor cells in the spleens of 0–3-day-old mice did not increase in response to antigenic stimulation, indicating that they too are unable at this stage to develop reactivity to immunological signals.     

Hepatoblast-like cells populate the adult p53 knockout mouse liver: evidence for a hyperproliferative maturation-arrested stem cell compartment.

Although p53 regulates the cell cycle and apoptosis, gross embryonic development is normal in the p53 knockout (-/-) mouse. In this study, we comprehensively assessed liver development in p53 -/- mice (from embryonic day 15 to adult) for evidence of a cell cycle-induced perturbation in differentiation. Liver cell proliferation in the embryo and newborn is similar in p53 -/- and +/+ mice; in contrast, -/- adult hepatocytes divide at twice the rate of wild types. Developmental expression patterns of liver-specific markers that are up-regulated (e.g., phosphoenolpyruvate carboxykinase and aldolase B) and down-regulated (e.g., alpha-fetoprotein) are similar. Therefore, embryonic and perinatal liver development is normal in the absence of p53. However, the p53 -/- adult liver displays small blast-like cells, the majority being hepatic and some lymphoid. These cells appear in periportal regions and can infiltrate the parenchyma. The hepatic blast-like cells express both mature and immature liver markers, suggesting that differentiation of the liver stem cell compartment is blocked.     

Induction of immune tolerance to thymus-dependent antigen (sheep erythrocytes) in the absence of T-cells]

The role of T cells in B cell tolerance induction to sheep red blood cells (SRBC) was studied in intact adult mice, in lethally irradiated mice injected with singeneic embryonic liver cells and thymocytes (TB-mice) and in animals functionally deprived of T cells--thymectomized, letally irradiated mice reconstituted with embryonic liver cells only (B-mice). Tolerance was obtained by treatment of mice with SRBC and cyclophosphamide (Cy). Cy-induced tolerance to SRBC was shown to be the result of the absence of specific T cells and partially of immunocompetent B cells. Suppression of immunoreactivity was observed not only in TB-mice but also in B-mice subjected to tolerogenic treatment. Splenocytes of tolerant TB-mice did not suppress the immune response of intact spleen cells to SRBC. The results obtained suggest the conclusion that B cells tolerance could be formed in absence of T cells.     

Neonatal susceptibility to MHV3 infection in mice. I. Transfer of resistance.

Up to 3 weeks of age, mice of the resistant A/J strain are fully susceptible to mouse hepatitis virus type 3 infection (MHV3). Immune deficiency, however, resulting from neonatal thymectomy or long term ALS administration led A/J animals to remain susceptible when tested at adult age. Whole spleen cells transferred from normal adult A/J donor mice protected suckling syngeneic recipients from i.p. infection with MHV3. Such a protective capacity of spleen cells was abolished after treatment with anti-theta serum and complement. Spleen cell separation by means of adherence to plastic also showed that neither the nonadherent nor the adherent populations injected separately were able to confer resistance to young mice challenged with the virus. Protection was not achieved with peritoneal cells originating from adult syngeneic animals. Transfer of resistance to MHV3 was obtained, however, when peritoneal cells were associated with adherent spleen cells. This study indicated that two types of mature cells, at least, were required for transferring MHV3 resistance into newborn mice of the A/J strain: T lymphocytes and an adherent spleen cell population.     

Fetal erythroid cell development: density gradients and size distributions

Electronic size distributions of erythroid cells from fetal C57BL-6J mice during the eleventh through twentieth days of gestation indicate that the erythropoietic cell populations are constantly changing. The mean volume of the liver derived non-nucleated erythroid population decreases from four times the mean adult erythrocyte volume on the thirteenth gestation day to twice the adult erythrocyte volume at birth. The mean volume of the nucleated erythroid cell is about ten times the mean adult erythrocyte volume. The gestation age of an embryo can be determined from blood cell size distributions. The mode of the non-nucleated population and the percentage of each population indicates the gestation age. Size distribution of cells in density gradient fractions apparently indicate two size populations of non-nucleated cells between the thirteenth and fifteenth days. The density of the non-nucleated cells increases during gestation. It is suggested that the decrease in size and increase in density of non-nucleated cells is due to the release of successively smaller reticulocytes from the liver.