Inflammatory biomarker, neopterin, suppresses B lymphopoiesis for possible facilitation of granulocyte responses, which is severely altered in age-related stromal-cell-impaired mice, SCI/SAM

Neopterin is produced by monocytes and is a useful biomarker of inflammatory activation. We found that neopterin enhanced in vivo and in vitro granulopoiesis triggered by the stromal-cell production of cytokines in mice. The effects of neopterin on B lymphopoiesis during the enhancement of granulopoiesis were determined using the mouse model of senescent stromal-cell impairment (SCI), a subline of senescence-accelerated mice (SAM). In non-SCI mice (a less senescent stage of SCI mice), treatment with neopterin decreased the number of colonies, on a semisolid medium, of colony-forming units of pre-B-cell progenitors (CFU-preB) from unfractionated bone marrow (BM) cells, but not that from a population rich in pro-B and pre-B cells without stromal cells. Neopterin upregulated the expression of genes for the negative regulators of B lymphopoiesis such as tumor necrosis factor-alpha (TNF-alpha ), interleukin-6 (IL-6), and transforming growth factor-beta (TGF-beta) in cultured stromal cells, implying that neopterin suppressed the CFU-preB colony formation by inducing negative regulators from stromal cells. The intraperitoneal injection of neopterin into non-SCI mice resulted in a marked decrease in the number of femoral CFU-preB within 1 day, along with increases in TNF-alpha and IL-6 expression levels. However, in SCI mice, in vivo and in vitro responses to B lymphopoiesis and the upregulation of cytokines after neopterin treatment were less marked than those in non-SCI mice. These results suggest that neopterin predominantly suppressed lymphopoiesis by inducing the production of negative regulators of B lymphopoiesis by stromal cells, resulting in the selective suppression of in vivo B lymphopoiesis. These results also suggest that neopterin facilitated granulopoiesis in BM by suppressing B lymphopoiesis, thereby contributing to the potentiation of the inflammatory process; interestingly, such neopterin function became impaired during senescence because of attenuated stromal-cell function, resulting in the downmodulation of the host-defense mechanism in the aged.     

The BiP Cochaperone ERdj4 Is Required for B Cell Development and Function

ERdj4 is a BiP cochaperone regulated by the unfolded protein response to facilitate degradation of unfolded and/or misfolded proteins in the endoplasmic reticulum. As the unfolded protein response plays a critical role in B cell maturation and antibody production, ERdj4 gene trap mice were generated to determine if this chaperone was required for B cell homeostasis. Homozygosity for the trapped allele resulted in hypomorphic expression of ERdj4 in bone marrow cells and abnormal development of hematopoietic lineages in the bone marrow. The number of myeloid cells was increased, while the number of erythroid and B lymphoid cells was reduced in ERdj4 gene trap mice compared to controls. An intrinsic B cell defect was identified that decreased survival of B cell precursors including large and small pre-B, and immature B cells. Consistent with impaired B lymphopoiesis, the number of mature follicular B cells was reduced in both the bone marrow and spleen of ERdj4 gene trap mice. Paradoxically, unchallenged ERdj4 gene trap mice showed non-specific hypergammaglobulinemia and gene trap B cells exhibited increased proliferation, survival and isotype switching in response to LPS stimulation. Although ERdj4 gene trap mice responded normally to T cell-independent antigen, they failed to mount a specific antibody response to T cell-dependent antigen in vivo. Collectively, these findings demonstrate that the chaperone activity of ERdj4 is required for survival of B cell progenitors and normal antibody production.     

Apurinic/apyrimidinic endonuclease 2 is necessary for normal B cell development and recovery of lymphoid progenitors after chemotherapeutic challenge

B cell development involves rapid cellular proliferation, gene rearrangements, selection, and differentiation, and it provides a powerful model to study DNA repair processes in vivo. Analysis of the contribution of the base excision repair pathway in lymphocyte development has been lacking primarily owing to the essential nature of this repair pathway. However, mice deficient for the base excision repair enzyme, apurinic/apyrimidinic endonuclease 2 (APE2) protein develop relatively normally, but they display defects in lymphopoiesis. In this study, we present an extensive analysis of bone marrow hematopoiesis in mice nullizygous for APE2 and find an inhibition of the pro-B to pre-B cell transition. We find that APE2 is not required for V(D)J recombination and that the turnover rate of APE2-deficient progenitor B cells is nearly normal. However, the production rate of pro- and pre-B cells is reduced due to a p53-dependent DNA damage response. FACS-purified progenitors from APE2-deficient mice differentiate normally in response to IL-7 in in vitro stromal cell cocultures, but pro-B cells show defective expansion. Interestingly, APE2-deficient mice show a delay in recovery of B lymphocyte progenitors following bone marrow depletion by 5-fluorouracil, with the pro-B and pre-B cell pools still markedly decreased 2 wk after a single treatment. Our data demonstrate that APE2 has an important role in providing protection from DNA damage during lymphoid development, which is independent from its ubiquitous and essential homolog APE1.     

Abnormalities of B lineage cells are demonstrable in long term lymphoid bone marrow cultures of New Zealand black mice

The formation of B lymphocytes in young New Zealand Black (NZB) mice proceeds at an accelerated rate resulting in a deficiency of B lineage precursors in adult (greater than 15 wk old) animals. To study the characteristics of B lineage cells in young (4 wk) and old (6 mo) NZB mice, bone marrow from these animals was used to initiate long term lymphoid bone marrow cultures (LBMC) that permit the long term maintenance of B cells and their precursors. Age-matched cultures from BALB/c mice and NZB.xid marrow were established in parallel. Primary LBMC were readily established from these strains and showed similar patterns of growth for the 3-mo observation period. No significant differences in numbers of 14.8 positive cells were observed. However, NZB mice at both ages had a higher percentage of membrane IgM (mIgM)-expressing cells. Significant levels of supernatant IgM were found only in cultures of 6-mo NZB and BALB/c mice; levels were highest in NZB culture supernatants and were often more than 500 ng/ml; significant, although much lower, levels of IgG were likewise detected. Lymphoid cells from NZB.xid mice were unable to generate significant levels of IgM in supernatant fluids indicating the effects of the xid gene were displayed in vitro. Autoantibodies were not detected in any of the culture supernatants. Additional evidence for NZB hyperactivity in primary B lymphopoiesis was observed upon initiation of primary myeloid bone marrow cultures (MBMC) from these strains of mice and subsequently transferring them to LBMC conditions. This results in the cessation of myelopoiesis at the initiation of B lymphopoiesis. At the time of converting MBMC to LBMC, cultures of NZB and BALB/c mice morphologically resembled myeloid cultures and had neither B cell colony-forming units nor cells that expressed 14.8 or mIgM. However, following the switch, NZB mice had a 5-fold higher number of B cell colony-forming units. Further, MBMC established from NZB bone marrow cells had a reduced capacity to form colonies in the granulocyte-macrophage colony-forming unit assay. These studies indicate that defects of NZB hemopoietic cells are manifest in vitro and suggest the use of in vitro long term cultures as a valuable technique to further dissect the hematopoietic abnormalities of NZB mice and possible underlying microenvironmental defects.     

In vitro differentiation of B lymphocytes from primitive hemopoietic precursors present in long-term bone marrow cultures

B lymphocytes are not produced in the Dexter long-term bone marrow cultures, but a primitive B cell precursor is present. The findings presented in this study demonstrate that this precursor can be induced to produce B lymphocytes by transferring the cultures to the Whitlock conditions for the long-term growth of B cells in vitro. Two weeks after the transfer of cultures maintained at 33 degrees C in medium supplemented with horse serum and steroids to low concentrations of fetal calf serum at 37 degrees C, marked effects can be observed. The pattern of cell growth changes from one in which the hemopoietic cells are clustered in tight foci containing several hundred cells to smaller ones in which the cells are not as densely packed. Fat cells in the adherent layer disappear and the supporting stroma becomes more uniform in appearance. This change in the culture format is accompanied by a decrease in the number of nonadherent cells and a shift from myelopoiesis to lymphopoiesis. The numbers of granulocyte-macrophage progenitors decline weekly after the change in culture conditions and are not detected by the third week. B cell colony-forming units appear by 3 wk. Cells that express the 14.8 cell surface antigen are induced by 1 wk after the change in culture conditions, followed by the appearance of surface IgM-bearing cells 2 wk later. This shift to lymphopoiesis can be confirmed morphologically. Granulocytes and macrophages disappear from the cultures by 4 wk, at which time almost all of the cells have a characteristic lymphocyte morphology. Upon switching these cultures back to the original Dexter conditions, only low levels of transient myelopoiesis can be reinitiated.     

B lymphocyte development in rabbit: progenitor B cells and waning of B lymphopoiesis

In mammals that use gut-associated lymphoid tissues for expansion and somatic diversification of the B cell repertoire, B lymphopoiesis occurs early in ontogeny and does not appear to continue throughout life. In these species, including sheep, rabbit, and cattle, little is known about the pathway of B cell development and the time at which B lymphopoiesis wanes. We examined rabbit bone marrow by immunofluorescence with anti-CD79a and anti-mu and identified both proB and preB cells. The proB cells represent the vast majority of B-lineage cells in the bone marrow at birth and by incorporation of 5-bromo-2'-deoxyuridine, they appear to be a dynamic population. PreB cells reach maximum levels in the bone marrow at 3 wk of age, and B cells begin to accumulate at 7 wk of age. We cloned two VpreB and one lambda5 gene and demonstrated that they are expressed within B-lineage cells in bone marrow. VpreB and lambda5 coimmunoprecipitated with the mu-chain in lysates of 293T cells transfected with VpreB, lambda5, and mu, indicating that VpreB, lambda5, and mu-chains associate in a preB cell receptor-like complex. By 16 wk of age, essentially no proB or preB cells are found in bone marrow and by PCR amplification, B cell recombination excision circles were reduced 200-fold. By 18 mo of age, B cell recombination excision circles were reduced 500- to 1000-fold. We suggest that B cell development in the rabbit occurs primarily through the classical, or ordered, pathway and show that B lymphopoiesis is reduced over 99% by 16 wk of age.     

A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis

The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.     

IL-3 increases production of B lymphoid progenitors from human CD34+CD38- cells

The effect of IL-3 on the B lymphoid potential of human hemopoietic stem cells is controversial. Murine studies suggest that B cell differentiation from uncommitted progenitors is completely prevented after short-term exposure to IL-3. We studied B lymphopoiesis after IL-3 stimulation of uncommitted human CD34+CD38- cells, using the stromal cell line S17 to assay the B lymphoid potential of stimulated cells. In contrast to the murine studies, production of CD19+ B cells from human CD34+CD38- cells was significantly increased by a 3-day exposure to IL-3 (p < 0.001). IL-3, however, did not increase B lymphopoiesis from more mature progenitors (CD34+CD38+ cells) or from committed CD34-CD19+ B cells. B cell production was increased whether CD34+CD38- cells were stimulated with IL-3 during cocultivation on S17 stroma, on fibronectin, or in suspension. IL-3Ralpha expression was studied in CD34+ populations by RT-PCR and FACS. High IL-3Ralpha protein expression was largely restricted to myeloid progenitors. CD34+CD38- cells had low to undetectable levels of IL-3Ralpha by FACS. IL-3-responsive B lymphopoiesis was specifically found in CD34+ cells with low or undetectable IL-3Ralpha protein expression. IL-3 acted directly on progenitor cells; single cell analysis showed that short-term exposure of CD34+CD38- cells to IL-3 increased the subsequent cloning efficiency of B lymphoid and B lymphomyeloid progenitors. We conclude that short-term exposure to IL-3 significantly increases human B cell production by inducing proliferation and/or maintaining the survival of primitive human progenitors with B lymphoid potential.     

Effects of aging on the common lymphoid progenitor to pro-B cell transition

The number of common lymphoid progenitors (CLP) and their pre-pro-B and pro-B cell progeny is reduced in old mice, but the age-related changes responsible for these declines have not been fully elucidated. The aim of this study was to provide additional insights into the impact of senescence on early B cell development by analyzing the CLP and pro-B cell compartments under steady-state conditions and after cytoablation with 5-fluorouracil. 5-Fluorouracil subjects the hemopoietic system to acute stress and has the advantage of revealing defects in progenitors that may otherwise be subtle. The data demonstrate significant, age-related defects in the proliferative potential of early B cell precursors and suggest that the ability of CLP to differentiate into pre-pro-B cells is also compromised by senescence. These age-related changes in early B lymphopoiesis do not result from a general defect in HSC or the bone marrow microenvironment that impairs development in all hemopoietic lineages. Instead, data demonstrating that myeloid progenitor number and developmental potential do not decline with age indicate that B lymphopoiesis is particularly sensitive to defects that accumulate during senescence.     

IL-7 induces myelopoiesis and erythropoiesis

IL-7 administration to mice was previously reported to increase the mobilization of progenitor cells from marrow to peripheral sites. We now report that IL-7 increases the number of mature myeloid and monocytic cells in spleen and peripheral blood. This effect required T cells, and we show that IL-7 treatment in vivo induced GM-CSF and IL-3 production by T cells with memory phenotype. However, additional myelopoietic cytokines were shown to be involved because mice deficient in both GM-CSF and IL-3 also responded to IL-7 with increased myelopoiesis. Candidate cytokines included IFN-gamma and Flt3 ligand, which were also produced in response to IL-7. Because IFN-gamma-deficient mice also increased myelopoiesis, it was suggested that IL-7 induced production of redundant myelopoietic cytokines. In support of this hypothesis, we found that the supernatant from IL-7-treated, purified T cells contained myelopoietic activity that required a combination of Abs against GM-CSF, IL-3, and anti-Flt3 ligand to achieve maximum neutralization. IL-7 administration increased the number of splenic erythroid cells in either normal, Rag1 or GM-CSF-IL-3-deficient mice, suggesting that IL-7 might directly act on erythroid progenitors. In support of this theory, we detected a percentage of TER-119(+) erythroid cells that expressed the IL-7Ralpha-chain and common gamma-chain. Bone marrow cells expressing IL-7R and B220 generated erythroid colonies in vitro in response to IL-7, erythropoietin, and stem cell factor. This study demonstrates that IL-7 can promote nonlymphoid hemopoiesis and production of cytokines active in the host defense system in vivo, supporting its possible clinical utility.     

Adiponectin, a fat cell product, influences the earliest lymphocyte precursors in bone marrow cultures by activation of the cyclooxygenase-prostaglandin pathway in stromal cells

Adiponectin, an adipocyte-derived hormone, is attracting considerable interest as a potential drug for diabetes and obesity. Originally cloned from human s.c. fat, the protein is also found in bone marrow fat cells and has an inhibitory effect on adipocyte differentiation. The aim of the present study is to explore possible influences on lymphohematopoiesis. Recombinant adiponectin strongly inhibited B lymphopoiesis in long-term bone marrow cultures, but only when stromal cells were present and only when cultures were initiated with the earliest category of lymphocyte precursors. Cyclooxygenase inhibitors abrogated the response of early lymphoid progenitors to adiponectin in stromal cell-containing cultures. Furthermore, PGE(2), a major product of cyclooxygenase-2 activity, had a direct inhibitory influence on purified hematopoietic cells, suggesting a possible mechanism of adiponectin action in culture. In contrast to lymphopoiesis, myelopoiesis was slightly enhanced in adiponectin-treated bone marrow cultures, and even when cultures were initiated with single lymphomyeloid progenitors. Finally, human B lymphopoiesis was also sensitive to adiponectin in stromal cell cocultures. These results suggest that adiponectin can negatively and selectively influence lymphopoiesis through induction of PG synthesis. They also indicate ways that adipocytes in bone marrow can contribute to regulation of blood cell formation.     

Osteoclast activity modulates B-cell development in the bone marrow

B-cell development is dependent on the interactions between B-cell precursors and bone marrow stromal cells, but the role of osteoclasts (OCLs) in this process remains unknown. B lymphocytopenia is a characteristic of osteopetrosis, suggesting a modulation of B lymphopoiesis by OCL activity. To address this question, we first rescued OCL function in osteopetrotic oc/oc mice by dendritic cell transfer, leading to a restoration of both bone phenotype and B-cell development. To further explore the link between OCL activity and B lymphopoiesis, we induced osteopetrosis in normal mice by injections of zoledronic acid (ZA), an inhibitor of bone resorption. B-cell number decreased specifically in the bone marrow of ZA-treated mice. ZA did not directly affect B-cell differentiation, proliferation and apoptosis, but induced a decrease in the expression of CXCL12 and IL-7 by stromal cells, associated with reduced osteoblastic engagement. Equivalent low osteoblastic engagement in oc/oc mice confirmed that it resulted from the reduced OCL activity rather than from a direct effect of ZA on osteoblasts. These dramatic alterations of the bone microenvironment were disadvantageous for B lymphopoiesis, leading to retention of B-cell progenitors outside of their bone marrow niches in the ZA-induced osteopetrotic model. Altogether, our data revealed that OCLs modulate B-cell development in the bone marrow by controlling the bone microenvironment and the fate of osteoblasts. They provide novel basis for the regulation of the retention of B cells in their niche by OCL activity.     

Granulocyte-macrophage colony-stimulating factor as an autocrine survival factor for mature normal and malignant B lymphocytes

The role of GM-CSF in B cell (patho)physiology is unclear. Although B cells can respond to GM-CSF, there is controversy concerning the extent to which various resting and activated B cell types can themselves produce this cytokine, and the possibility that it can function in an autocrine fashion has not previously been considered. The aim of the present study was to address these issues using hairy cells (HCs) and chronic lymphocytic leukemia cells, two intrinsically activated mature malignant B cell types (with activation being more uniform and more pronounced in HCs). Normal B cells were used for comparison. Using a number of techniques, we demonstrated the constitutive production of GM-CSF by all three cell types and showed that the cytokine was biologically active. GM-CSF mRNA and protein were increased after cell activation by PMA, and constitutive production of the cytokine was highest in HCs, suggesting that the level of GM-CSF production is influenced by cell activation. Because GM-CSF is known to be antiapoptotic for myeloid cells, we used blocking anti-GM-CSF Abs to examine the contribution of autocrinely produced cytokine to cell survival. The Abs produced marked reduction in the in vitro survival of HCs, chronic lymphocytic leukemia cells, and normal B cells by promoting apoptosis. Taken together, these findings suggest that, in combination with other known rescue factors, autocrinely produced GM-CSF may contribute to normal and malignant B cell survival in vivo.     

Increased and long-term generation of dendritic cells with reduced function from IL-6-deficient bone marrow

The importance of IL-6 in dendritic cell (DC) development and function has not been well defined. To establish the role of IL-6, we studied bone marrow-derived DC (BMDC) and freshly isolated splenic DC from IL-6(-/-)-transgenic mice. We found that although IL-6(-/-) bone marrow had a similar composition to that of wild-type (WT) mice, it generated up to 10 times more DC when cultured in GM-CSF. The difference persisted even when IL-6(-/-) and WT bone marrow were cultured together, excluding the possibility that the effects were simply due to different cytokine microenvironments. In comparison to WT BMDC, IL-6(-/-) BMDC captured at least as much Ag, had an equivalent surface phenotype, and matured similarly in response to LPS or CpG. However, IL-6(-/-) BMDC induced less T cell allostimulation and Ag-specific T cell activation, but only the former was related to their inability to generate IL-6. Although WT bone marrow cultures died within 4 wk, IL-6(-/-) cultures continued to generate BMDC for >120 days, although the BMDC became immature and less functional. In vivo, we found that IL-6(-/-) mice had similar numbers and types of splenic DC as WT mice, both normally and after treatment with either Flt-3 ligand or GM-CSF. These findings demonstrate that IL-6 has profound effects on DC development in vitro, although the number and subtype composition of DC are unaffected by the absence of IL-6 in vivo. Furthermore, secretion of IL-6 is critical to certain DC functions.     

Effects of liver-derived dendritic cell progenitors on Th1- and Th2-like cytokine responses in vitro and in vivo

There is evidence that donor-derived dendritic cells (DC), particularly those at a precursor/immature stage, may play a role in the immune privilege of liver allografts. Underlying mechanisms are poorly understood. We have examined the influence of in vitro generated mouse liver-derived DC progenitors (DCp) on proliferative, cytotoxic, and Th1/Th2 cytokine responses induced in allogeneic T cells. Liver DCp, propagated in GM-CSF from C57B10 mice (H2b), induced only minimal proliferation, and weak cytotoxic responses in allogeneic (C3H; H2k) T cells compared with mature bone marrow (BM)-derived DC. Flow-cytometric analysis of intracellular cytokine staining revealed that mature BM DC, but not liver DCp, elicited CD4+ T cell production of IFN-gamma. Intracellular expression of IL-10 was very low in both BM DC- and liver DCp-stimulated CD4+ T cells. Only stimulation by liver DCp was associated with IL-10 secretion in primary MLR. Notably, these liver DCp cocultured with allogeneic T cells stained strongly for IL-10. Following local (s.c. ) injection in allogeneic recipients, both BM DC and liver DCp homed to T cell areas of draining lymph nodes and spleen, where they were readily detected by immunohistochemistry up to 2 wk postinjection. Liver DCp induced clusters of IL-10- and IL-4-secreting mononuclear cells, whereas Th2 cytokine-secreting cells were not detected in mice injected with mature BM DC. By contrast, comparatively high numbers of IFN-gamma+ cells were induced by BM DC. Modulation of Th2 cytokine production by donor-derived DCp may contribute to the comparative immune privilege of hepatic allografts.     

Age-dependent changes in B lymphocyte lineage cell populations of autoimmune-prone BXSB mice

BXSB mice, a recently developed autoimmune strain, develop a human lupus-like disease with B cell hyperplasia in peripheral lymphoid organs. Unlike other experimental models of autoimmunity and human lupus, BXSB male mice manifest accelerated autoimmune phenomena through the influence of a Y chromosome-linked enhancing factor. The present studies were performed to investigate the features of B lymphopoiesis in BXSB mice and to determine whether differences exist between BXSB males and females in this respect. B lineage cell populations in the marrow of BXSB mice were identified phenotypically by studying the cytoplasmic mu-heavy chains of IgM (c mu), and functionally by their ability to acquire clonability and sIg in short-term liquid cultures. Male BXSB mice became deficient in both the precursors of functional B cells and c mu + pre-B cells by the age of 8 to 12 wk. This followed a transient increase in this population, which peaked when the mice were 2 to 4 wk old. In females, substantial numbers of functional B cell precursors and c mu + cells were maintained until more than 4 mo of age. Cells lacking Ig but bearing a B lineage cell antigen (14.8) were elevated in numbers in both BXSB males and females until 16 wk of age when compared to normal strains of mice. At the time pre-B cells and functional B precursors were elevated in numbers, some sIg- cells were shown to form colonies in mitogen-stimulated semisolid agar cultures without a period of preculture. Most of these sIg- cells seemed to bear the B lineage cell antigen (14.8). They were independent of both G-10 adherent regulatory cells and Thy-1+ cells for their colony formation. These results indicate that B lymphocyte formation may be maintained in a hyperactive state in BXSB females, whereas males become deficient in B cell precursors very early in life. This early decline might be related to the accelerated development of autoimmune disease in BXSB mice. Bone marrow transplantation studies showed that these unusual characteristics of B lymphopoiesis were reciprocally transferable with unseparated bone marrow cells between BXSB males and females. This finding indicates that sex hormones are not a critical variable in abnormal B lymphocyte formation in this strain, and that the premature deficiency of immediate B precursors in males may be regulated by a genetic factor(s) located on the Y chromosome.     

Increased generation of dendritic cells from myeloid progenitors in autoimmune-prone nonobese diabetic mice

Aberrant dendritic cell (DC) development and function may contribute to autoimmune disease susceptibility. To address this hypothesis at the level of myeloid lineage-derived DC we compared the development of DC from bone marrow progenitors in vitro and DC populations in vivo in autoimmune diabetes-prone nonobese diabetic (NOD) mice, recombinant congenic nonobese diabetes-resistant (NOR) mice, and unrelated BALB/c and C57BL/6 (BL/6) mice. In GM-CSF/IL-4-supplemented bone marrow cultures, DC developed in significantly greater numbers from NOD than from NOR, BALB/c, and BL/6 mice. Likewise, DC developed in greater numbers from sorted (lineage(-)IL-7Ralpha(-)SCA-1(-)c-kit(+)) NOD myeloid progenitors in either GM-CSF/IL-4 or GM-CSF/stem cell factor (SCF)/TNF-alpha. [(3)H]TdR incorporation indicated that the increased generation of NOD DC was due to higher levels of myeloid progenitor proliferation. Generation of DC with the early-acting hematopoietic growth factor, flt3 ligand, revealed that while the increased DC-generative capacity of myeloid-committed progenitors was restricted to NOD cells, early lineage-uncommitted progenitors from both NOD and NOR had increased DC-generative capacity relative to BALB/c and BL/6. Consistent with these findings, NOD and NOR mice had increased numbers of DC in blood and thymus and NOD had an increased proportion of the putative myeloid DC (CD11c(+)CD11b(+)) subset within spleen. These findings demonstrate that diabetes-prone NOD mice exhibit a myeloid lineage-specific increase in DC generative capacity relative to diabetes-resistant recombinant congenic NOR mice. We propose that an imbalance favoring development of DC from myeloid-committed progenitors predisposes to autoimmune disease in NOD mice.     

B cell maintenance in aged mice reflects both increased B cell longevity and decreased B cell generation

In aged mice the population of mature peripheral B cells is maintained despite a severalfold decrease in the population of bone marrow B cell progenitors. The analysis of the rate of accumulation of 5'-bromo-2-deoxyuridine (BrdU)-labeled splenic B cells in mice fed BrdU for 8 days to 8 wk demonstrated a severalfold increase in the half-life of mature B cells in aged mice. Consistent with a role for decreased B cell turnover in maintaining the mature B cell population of aged mice, several findings indicate that fewer newly generated B cells enter the spleen from the bone marrow in aged vs young adult mice. These include 1) a fourfold decrease in the population of relatively immature splenic B cells, defined as cells that express high levels of heat-stable Ag and accumulate BrdU within 8 wk of labeling; and 2) an equivalent decrease in the population of bone marrow cells representative of later stages of B cell maturation (sIgD-sIgM(int-high)). Surprisingly, despite a four- to sixfold decrease in pre-B cells, the population of least mature bone marrow B cells (IgD-sIgM(very low)) remains intact. Because this population accumulates BrdU-labeled cells more slowly in aged mice than in younger mice, and bone marrow B cells at more mature developmental stages are diminished, it appears that in aged mice B cell development beyond the sIgM(very low) stage may be retarded and that cells, therefore, accumulate within this population.     

Lidocaine depresses splenocyte immune functions following trauma-hemorrhage in mice

Traumatic and/or surgical injury as well as hemorrhage induces profound suppression of cellular immunity. Although local anesthetics have been shown to impair immune responses, it remains unclear whether lidocaine affects lymphocyte functions following trauma-hemorrhage (T-H). We hypothesized that lidocaine will potentiate the suppression of lymphocyte functions after T-H. To test this, we randomly assigned male C3H/HeN (6–8 wk) mice to sham operation or T-H. T-H was induced by midline laparotomy and 90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (4x shed blood volume in the form of Ringer lactate). Two hours later, the mice were killed and splenocytes and bone marrow cells were isolated. The effects of lidocaine on concanavalin A-stimulated splenocyte proliferation and cytokine production in both sham-operated and T-H mice were assessed. The effects of lidocaine on LPS-stimulated bone marrow cell proliferation and cytokine production were also assessed. The results indicate that T-H suppresses cell proliferation, Th1 cytokine production, and MAPK activation in splenocytes. In contrast, cell proliferation, cytokine production, and MAPK activation in bone marrow cells were significantly higher 2 h after T-H compared with shams. Lidocaine depressed immune responses in splenocytes; however, it had no effect in bone marrow cells in either sham or T-H mice. The enhanced immunosuppressive effects of lidocaine could contribute to the host's enhanced susceptibility to infection following T-H. shock; bone marrow cells     

The role of endogenous glucocorticoids in lymphocyte development in melanocortin receptor 2-deficient mice

Glucocorticoids are extensively used in anti-inflammatory therapy and are thought to contribute to the steady-state regulation of hematopoiesis and lymphopoiesis. We have previously established MC2R(-/-) mice, a model of familial glucocorticoid deficiency, that show several similarities to patients with this disease, including undetectable levels of corticosterone, despite high levels of ACTH and unresponsiveness to ACTH. In this study, we analyzed the possible roles of endogenous glucocorticoids in hematopoiesis and lymphopoiesis in MC2R(-/-) and CRH(-/-) mice as models of chronic adrenal insufficiency. Our analysis of total peripheral blood cell counts revealed that the number of lymphocytes was increased and the number of erythrocytes was slightly, but significantly, decreased in MC2R(-/-) mice. Numbers of immature double negative (CD4(-) CD8(-)) thymocytes, transitional type 1 B cells in the spleen, and pre-B cells in the bone marrow, were significantly increased in MC2R(-/-) mice, suggesting that endogenous glucocorticoids contribute to steady-state regulation of lymphopoiesis. Oral glucocorticoid supplementation reversed peripheral blood cell counts and reduced numbers of T and B cells in the thymus and the spleen. T cells in the thymus and B cells in the spleen were also increased in CRH(-/-) mice, another animal model of chronic adrenal insufficiency. MC2R(-/-) mice were sensitive to age-related thymic involution, but they were resistant to fasting-associated thymic involution. Our data support the idea that endogenous glucocorticoids contribute to stress-induced as well as steady-state regulation of hematopoiesis and lymphopoiesis.