Bone marrow stromal cells mediate androgenic suppression of B lymphocyte development

Castration of normal male mice induces expansion of the bone marrow B cell population, an effect that can be reversed by androgen replacement. We employed in vitro cultures and two in vivo models to investigate whether androgens exert these effects directly on marrow lymphoid precursors or whether actions on marrow stromal elements are required. Immature B cells from normal mouse bone marrow were not responsive to the suppressive effect of androgens unless they were cocultured with marrow stromal cells or with supernatants from androgen-treated stromal cells, suggesting that the androgen effects are exerted through marrow stromal elements by production of a diffusible mediator. Further experiments revealed that bone marrow stromal cells produced TGF-beta in response to dihydrotestosterone (DHT), and neutralization of TGF-beta in the DHT-treated stromal cells reversed the suppressive effects. The stromal cell requirement for androgen-mediated effects was confirmed in vivo by experiments using chimeric animals created by bone marrow transplantation in which androgen receptor expression was restricted to either the stromal or lymphoid cells of the bone marrow. Androgens only affected B cell development in chimeric mice with androgen-sensitive stromal cells. These experiments suggest that effects of androgens on developing B cells are mediated through androgen receptors in bone marrow stromal cells. TGF-beta is a candidate mediator for these hormonal effects.     

THE EFFECT OF HETEROLOGOUS ANTI-LYMPHOCYTE SERUM ON MOUSE HEMOPOIETIC STEM CELLS

The present study has demonstrated that rabbit anti-mouse lymphocyte serum (RAMLS) has the capability of destroying bone marrow cells and suppressing hemopoietic stem cell function. The in vitro incubation of bone marrow suspensions with RAMLS caused extensive cell lysis with an apparent preferential destruction of lymphoid, erythroid, and blastoid elements. Using the spleen colony assay, the number of functional hemopoietic stem cells was found to be markedly reduced in bone marrow populations exposed to RAMLS in vitro. Further, this loss of stem cell function could be produced by exposing marrow suspensions to small concentrations of antiserum which did not produce detectable cytotoxic effects on the general marrow population. A similar effect of RAMLS upon hemopoietic stem cells was found in vivo. The intravenous injection of RAMLS into lethally irradiated mice immediately after the infusion of isogeneic marrow cells reduced the number of spleen colonies formed, indicating that the antiserum could exhibit a deleterious effect upon stem cells in the bloodstream of the intact animal. Normal animals treated with daily subcutaneous injections of RAMLS for 3 wk had a significantly reduced marrow content of functional hemopoietic stem cells, suggesting that RAMLS can affect stem cells located in situ in the bone marrow. The experiments indicate that RAMLS possesses potential marrow toxicity.     

Similar rates of production of T and B lymphocytes in the bone marrow

The rate of renewal of T lymphocytes in the bone marrow of euthymic C57BL/Ka and athymic nu/nu BALB/c mice was estimated by in vivo labeling with bromodeoxyuridine. T lymphocytes accounted for 16-18% of marrow cells in euthymic mice as judged by immunofluorescent staining with monoclonal antibodies for Thy-1, CD3, and alpha/beta T cell antigen receptor markers. About 70% of marrow cells expressed receptors (Mac-1, Gr-1, B220) for myeloid, macrophage, and B lineage cells. Approximately 13% of cells in the athymic bone marrow expressed alpha/beta T cell receptors. Sorted marrow T cells proliferated in response to stimulation with anti-alpha/beta antibodies in vitro and showed functional rearrangements of V beta and J beta genes. Sorted non- T cells did not respond to stimulation in vitro, and all V beta and J beta gene rearrangements identified were nonfunctional. In vivo labeling studies indicated that approximately 17 x 10(6) bone marrow T cells are renewed daily in euthymic mice and approximately 14 x 10(6) are renewed in athymic mice. Approximately 11 x 10(6) mature B cells (immunoglobulin M+) are renewed daily in the bone marrow of the latter mice. To determine whether marrow precursors can give rise to T cells directly, marrow cells from euthymic and athymic mice were depleted of T cells by cell sorting and incubated in vitro for 48 h in the absence of exogenous growth factors or thymic stromal cells. Examination of the cells after culture showed that 10-12% stained brightly for alpha/beta T cell receptors. Although functional rearrangements of V beta and J beta genes were not detected before culture, the majority of rearrangements were functional after culture. The emergence of the bright alpha/beta T cells in culture was dependent on depletion T cells from the marrow cells before culture. The results suggest that most marrow T cells are generated in the marrow itself.     

The 86-kD subunit of Ku autoantigen mediates homotypic and heterotypic adhesion of multiple myeloma cells.

Previous studies have shown that triggering multiple myeloma (MM) cells via CD40 induces IL-6-mediated autocrine growth as well as increased expression of cell surface adhesion molecules including CD11a, CD11b, CD11c, and CD18. In this study, we generated the 5E2 mAb which targets an antigen that is induced upon CD40 ligand (CD40L) activation of MM cells. Immunofluorescence, immunoprecipitation, and protein sequencing studies identified the target antigen of 5E2 mAb as the 86-kD subunit of the Ku autoantigen. We demonstrate that increased cell surface expression of Ku on CD40L-treated cells is due to migration of Ku from the cytoplasm to the cell surface membrane. Moreover, cell surface Ku on CD40L-treated MM cells mediates homotypic adhesion of tumor cells, as well as heterotypic adhesion of tumor cells to bone marrow stromal cells and to human fibronectin; and 5E2 mAb abrogates IL-6 secretion triggered by tumor cell adherence to bone marrow stromal cells. These data suggest that CD40L treatment induces a shift of Ku from the cytoplasm to the cell surface, and are the first to show that Ku functions as an adhesion molecule. They further suggest that cell surface Ku may play a role in both autocrine and paracrine IL-6-mediated MM cell growth and survival.     

Characterization and cloning of a novel glycoprotein expressed by stromal cells in T-dependent areas of peripheral lymphoid tissues

A novel glycoprotein (gp) expressed by stromal cells of peripheral lymphoid tissue has been characterized immunohistochemically, biochemically, and at the molecular level. This molecule, gp38, was identified with a monoclonal antibody (mAb) (clone 8.1.1) previously shown to react with a subpopulation of thymic epithelium. This mAb generated a reticular labeling pattern in medullary and paracortical areas of lymph nodes and in splenic white pulp. At the ultrastructural level, labeling by the 8.1.1 mAb was restricted to fibroblastic reticular stromal cells. Serial sections of lymph node and spleen labeled with anti-CD3, anti-B220, and 8.1.1 mAbs clearly showed that the 8.1.1+ cells were associated with T cell-dependent areas. In severe combined immunodeficiency (SCID) or Nu/Nu mice, splenic white pulp also exhibited reticular labeling with the 8.1.1 mAb in the absence of detectable numbers of T cells, indicating that the appearance of 8.1.1-reactive stromal cells in discrete areas of peripheral lymphoid tissue was T cell independent. The cDNA encoding this stromal cell molecule was cloned by direct expression in COS cells and found to encode a 172 amino acid sequence with the typical features of a type I integral membrane protein. COS cells transfected with the gp38 clone direct the expression of an approximately 38-kD protein that reacts with the 8.1.1 mAb but not with isotype-matched controls. Comparison of the predicted amino acid sequence of 8.1.1 mAb but not with isotype-matched controls. Comparison of the predicted amino acid sequence of 8.1.1 with proteins in the National Biomedical Research Foundation (NBRF) data base showed that gp38 is very closely related to the early response protein OTS-8 obtained from a cDNA library of tumor promoting agent (TPA)-induced murine osteoblastic cell line, MC3T3-E1.     

Committed T lymphocyte stem cells of rats. Characterization by surface W3/13 antigen and radiosensitivity

The existence of stem cells committed to the T lymphoid lineage was deduced from studying how rat T and B stem cells differ in their expression of membrane W3/13 antigen and in their susceptibility in vivo to gamma irradiation. Stem cell activity of rat bone marrow and fetal liver was measured in long-term radiation chimeras using B and T cell alloantigenic surface markers to identify the progeny of donor cells. Monoclonal mouse anti-rat thymocyte antibody W3/13 labeled approximately 40% of fetal liver cells and 60-70% of young rat bone marrow cells (40% brightly, 25% dimly). Bright, dim, and negative cells were separated on a fluorescence-activated cell sorter. All B and T lymphoid stem cells in fetal liver were W3/13 bright, as were B lymphoid stem cells in bone marrow. W3/13 dim bone marrow had over half the T cell repopulating activity of unseparated marrow but gave virtually no B cell repopulation. In further experiments, the radiosensitivity of endogenous B and T lymphoid stem cells was determined by exposing host rats to between 4.5 and 10 Gy of gamma irradiation before repopulation with genetically marked marrow. The results depended on whether chimerism was assayed before day 50 or after day 100. At early times, a radioresistant T stem cell was indicated, whose activity waned later. Thus committed T stem cells of rats carry moderate amounts of W3/13 antigen and are more radioresistant but less permanently chimeragenic than the stem cells that regenerate B lymphocytes.     

Inhibition of collagen deposition in the extracellular matrix prevents the establishment of a stroma supportive of hematopoiesis in long-term murine bone marrow cultures.

Long-term production of murine hematopoietic cells in vitro is dependent on establishment of a complex microenvironment consisting of a variety of stromal cells and an extensive extracellular matrix which includes collagen, fibronectin, laminin, proteoglycans, and other undefined components adherent to the culture dishes. Cis-4-hydroxyproline (CHP), a relatively specific inhibitor of collagen secretion, was used to examine the role of extracellular collagen deposition in supporting hematopoiesis in long-term C57B1/6J mouse bone marrow cell cultures. Throughout the 10-wk culture period, all culture dishes contained either 0, 10, 25, or 50 micrograms/ml of CHP. All medium and nonadherent cells were removed at weekly intervals and replaced with fresh medium containing the previous concentrations of CHP. Nonadherent cells were assayed weekly for total cells and pluripotent, erythroid, megakaryocytic, and granulocytic-macrophage progenitor cells. Dishes were killed at selected intervals to assess protein and collagen synthesis in the adherent layer. Adherent cell numbers, as judged by microscopic examination and DNA assays, correlated inversely with CHP concentrations used and paralleled degree of collagen synthesis inhibition. The decreased hemopoietic progenitor cell production correlated closely with percent inhibition of collagen synthesis and stromal cellularity. The CHP concentrations tested were not directly toxic to hemopoietic progenitor cells. These studies demonstrate that collagen deposition in the extracellular matrix of murine bone marrow cell cultures is essential to the establishment of a functional stromal microenvironment that is supportive of long-term hematopoiesis.     

Identification of a 107-kD glycoprotein that mediates adhesion between stromal cells and hematolymphoid cells

The mechanism of cell complex formation between lymphocytes and stromal cells was investigated. We found that lymphoid lines of both T and B lineages could form cell complexes with stromal cells from the thymus as well as bone marrow but not with macrophages or typical fibroblast lines. Formation of these cell complexes is temperature dependent and requires the presence of Mg2+, active cellular metabolism, and microfilament assembly of cytoskeleton. We raised an antiserum against a thymic stromal cell clone (BATE-2) in rats and found that, after absorption, this serum could effectively block cell complex formation between lymphocytes and stromal cells from both thymus and bone marrow. An efficient blocking was obtained only when the antiserum was added at the initial stage of cell interaction. From the blocking experiments and the SDS-PAGE analysis of immunoprecipitated materials from the stromal cell surface, we identified a unique 107-kD glycoprotein on the stromal cells as a molecule for mediating stromal cell-lymphocyte interaction. This is further supported by the findings that an antiserum raised in hamsters against the excised gel band corresponding to 107 kD, which specifically immunoprecipitated the 107-kD molecule, effectively blocked the lymphocyte-stromal cell interaction. The possible function of this molecule in hematolymphoid development is discussed.     

Developmental potential of the earliest precursor cells from the adult mouse thymus.

A new, numerically minute population of cells representing the earliest T precursor cells in the adult mouse thymus has recently been isolated. This population has been shown to be similar to bone marrow hemopoietic stem cells in surface antigenic phenotype and to express moderate levels of CD4. We now show, by fluorescence-activated cell sorting and intrathymic transfer to irradiated mice, that this apparently homogeneous population differs from multipotent stem cells in expressing the surface stem cell antigen 2 (Sca-2), that it differs from most early B lineage cells in lacking B220 and class II major histocompatibility complex expression, and that it binds rhodamine 123 like an activated rather than a quiescent cell. Irradiated recipient mice differing at the Ly 5 locus were used to compare the developmental potential of these early intrathymic precursors with bone marrow stem cells. Only T lineage product cells were detected when the intrathymic precursor population was transferred back into an irradiated thymus. However, when the intrathymic precursor population was transferred intravenously, it displayed the capacity to develop into both B and T lymphoid cells in recipient bone marrow, spleen, and lymph nodes, but no donor-derived myeloid cells were detected. The absence of myeloid and erythroid precursor activity was confirmed by showing that the intrathymic precursor population was unable to develop into myeloid or erythroid spleen colonies on intravenous transfer or to form colonies in an agar culture. These findings indicate that this earliest intrathymic precursor population has become restricted (or strongly biased) to lymphoid lineage development, but not exclusively to T lymphocytes.     

Role of RANK ligand in mediating increased bone resorption in early postmenopausal women

Studies in rodents have implicated various cytokines as paracrine mediators of increased osteoclastogenesis during estrogen deficiency, but increases in RANKL, the final effector of osteoclastogenesis, have not been demonstrated. Thus, we isolated bone marrow mononuclear cells expressing RANKL on their surfaces by two-color flow cytometry using FITC-conjugated osteoprotegerin-Fc (OPG-Fc-FITC) as a probe. The cells were characterized as preosteoblastic marrow stromal cells (MSCs), T lymphocytes, or B lymphocytes by using Ab's against bone alkaline phosphatase (BAP), CD3, and CD20, respectively, in 12 premenopausal women (Group A), 12 early postmenopausal women (Group B), and 12 age-matched, estrogen-treated postmenopausal women (Group C). Fluorescence intensity of OPG-Fc-FITC, an index of the surface concentration of RANKL per cell, was increased in Group B over Groups A and C by two- to threefold for MSCs, T cells, B cells, and total RANKL-expressing cells. Moreover, in the merged groups, RANKL expression per cell correlated directly with the bone resorption markers, serum C-terminal telopeptide of type I collagen and urine N-telopeptide of type I collagen, in all three cell types and inversely with serum 17beta-estradiol for total RANKL-expressing cells. The data suggest that upregulation of RANKL on bone marrow cells is an important determinant of increased bone resorption induced by estrogen deficiency.     

Characterization of a new monoclonal antibody 6G7 that recognizes a unique antigen on myeloid and lymphoid cells

We generated a monoclonal antibody (mAb) 6G7, which recognizes a 220-kD antigen on selected subpopulations of normal myeloid and lymphoid cells and their malignant counterparts. 6G7 reacts with 90-95% of peripheral blood B cells, 70-80% of CD8(+) cells, 30-35% of CD4(+) cells, 20-40% of monocytes, and 20-40% of CD34(+) cells from bone marrow. 6G7 reacts with leukemic blasts in acute myeloid leukemia (14/16), adult acute lymphoblastic leukemia (ALL) (5/5), pediatric ALL (5/9), chronic lymphocytic leukemia (8/8), follicular lymphoma (7/7), and Burkitt's lymphoma (1/1). Long-term bone marrow culture of 6G7(+/-) cells showed the majority of clonogenic hematopoietic cells were in mAb 6G7 subpopulation. An immunotoxin of 6G7 and ricin A chain was cytotoxic to 6G7(+) leukemia cell lines. mAb 6G7 has potential clinical applications for targeted immunotherapy of both leukemia and lymphoma.     

The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems

The precise relationship between the stem cells for the lymphoid system and those for the blood-forming system is unclear. While it is generally assumed that the hemopoietic stem cell, the spleen colony-forming unit (CFU-S), is also the stem cell for the lymphoid system, there is little evidence for this hypothesis. To investigate the stem cells in these two systems, we irradiated bone marrow cells to induce unique chromosome aberrations in the stem cell population and injected them at limiting dilution into stem cell-deficient recipients. Several months (between 3 and 11) were allowed for the injected cells to repopulate the hemopoietic system. At that time, the bone marrow, spleen, and thymus were examined for a high frequency of cells having the same unique chromosome aberration. The presence of such markers shows that the marker was induced in a cell with extensive proliferative capacity, i.e., a stem cell. In addition, the splenic lymphocytes were stimulated with phytohemagglutinin (PHA) or lipopolysaccharide (LPS) to search for unique chromosomes in dividing T and B cells, respectively. Finally, bone marrow cells were injected into secondary irradiated recipients to determine if the marker occurred in CFU-S and to determine whether or not the same tissue distributions of marked cells could be propogated by bone marrow cells in a second recipient. After examination of 28 primary recipients, it was possible to identify three unique patterns of stem cell regeneration. In one set of mice, a unique chromosome marker was observed in CFU-S and in PHA- and LPS-stimulated cultures. These mice provide direct evidence for a pluripotent stem cell in bone marrow. In addition, two restricted stem cells were identified by this analysis. In three recipients, abnormal karyotypes were found only in myeloid cells and not in B and T lymphocytes. These mice presumably received a marked stem cell restricted to differentiate only into myeloid progeny. In three other recipients, chromosome aberrations were found only in PHA-stimulated cells; CFU-S and cells from LPS cultures did not have cells with the unique chromosome. This pattern suggests that bone marrow contains cells committed to differentiation only into T lymphocytes. For each of the three types of stem cells, secondary recipients had the same cellular distribution of marked cells as the primary recipients. This observation provides further evidence that unique markers can be induced in both pluripotent and restricted stem cells.     

Association of alkaline-phosphatase-positive reticulum cells in bone marrow with granulocytic precursors

In the bone marrow, an elaborate stroma forms the structural basis of the hemopoietic microenvironment. In this study, two different types of stromal cells were identified with certainty on tissue sections of intact bone marrow of rats and mice using light and electron microscopic histochemistry: (a) a fibroblast-type of reticulum cell which is characterized by having alkaline phosphatase associated with its plasma membrane. We refer to this cell as the alkaline-phosphatase- positive reticulum cell (Al-RC). It is closely associated with granulocytic precursors, particularly myeloblasts and neutrophilic promyelocytes. These reticulum cells may be found throughout the marrow but are concentrated near the endosteum. (b) a macrophage-type of reticulum cell which is characterized by its abundance of lysosomal acid phosphatase and is mainly associated with erythroid precursors (as observed by others). In contrast to the above-mentioned cell type, this latter cell was found to be distributed uniformly throughout the marrow. We speculate that the Al-RC are mesenchymal stromal cells necessary for granulocytic differentiation in bone marrow.     

Expression and function of c-kit in hemopoietic progenitor cells

The expression and function of a receptor tyrosine kinase, c-kit, in the adult bone marrow of the mouse were investigated by using monoclonal antibodies (mAbs) against the extracellular domain of murine c-kit. In adult C57BL/6 mouse, 7.8% of total bone marrow cells express c-kit on their surface. Half of the c-kit+ cells do not express lineage markers including Mac-1, Gr-1, TER-119, and B220, while the remainder coexpress myeloid lineage markers such as Mac-1 and Gr-1. After c-kit+ cells were removed from the bone marrow cell preparation, hemopoietic progenitor cells reactive to IL-3, GM-CSF, or M-CSF and also those which give rise to spleen colonies in irradiated recipients disappeared almost completely. Thus, most hemopoietic progenitors in the adult bone marrow express c-kit. To investigate whether or not c-kit has any role in the hemopoiesis of adult bone marrow, we took the advantage of one of the anti-c-kit mAbs that can antagonize the function of c-kit. As early as two days after the injection of 1 milligram of an antagonistic antibody, ACK2, almost all hemopoietic progenitor cells disappeared from the bone marrow, which eventually resulted in the absence of mature myeloid and erythroid cells in the bone marrow. These results provide direct evidence that c-kit is an essential molecule for constitutive intramarrow hemopoiesis, especially for the self-renewal of hemopoietic progenitor cells at various stages of differentiation.     

Immunologic and hematopoietic effects of CD40 stimulation after syngeneic bone marrow transplantation in mice.

CD40 is a molecule present on multiple cell types including B lymphocyte lineage cells. CD40 has been shown to play an important role in B cell differentiation and activation in vitro, although little is known concerning the effects of CD40 stimulation in vivo. We therefore examined the effects of CD40 stimulation in mice using a syngeneic bone marrow transplantation (BMT) model in an effort to augment B cell recovery after high dose therapy with hematopoietic reconstitution. After the BMT, mice were treated with or without 2-6 microg of a soluble recombinant murine CD40 ligand (srmCD40L) given intraperitoneally twice a week. A significant increase in B cell progenitors (B220+/ surface IgM-) was observed in the bone marrow of mice receiving the srmCD40L. The treated recipients also demonstrated improved B-cell function with increases in total serum immunoglobulin and increased splenic mitogen responsiveness to LPS being noted. Additionally, srmCD40L treatment promoted secondary lymphoid organ repopulation, accelerating germinal center formation in the lymph nodes. Total B cell numbers in the periphery were not significantly affected even with continuous srmCD40L administration. Lymphocytes obtained from mice treated with the ligand also had increases in T cell mitogen and anti-CD3 mAb responsiveness and acquired the capability to produce IL-4. Surprisingly, treatment with srmCD40L also produced hematopoietic effects in mice, resulting in an increase of BM and splenic hematopoietic progenitor cells in the mice after BMT. Treatment with srmCD40L significantly increased granulocyte and platelet recovery in the peripheral blood. Incubation of BMC with srmCD40L in vitro also resulted in increased progenitor proliferation, demonstrating that the hematopoietic effects of the ligand may be direct. Thus, stimulation of CD40 by its ligand may be beneficial in accelerating both immune and hematopoietic recovery in the setting of bone marrow transplantation.     

Role of the stroma of the hematopoietic organs in the development of hematologic diseases. Intramedullary regulation of granulomonocytopoiesis

Bone marrow trepanobiopsies were cultivated in the agar drop-liquid medium system. A total of 155 subjects with different diseases of the blood system and 31 controls were examined. It was shown that prolonged cultivation of trepanobiopsies makes it possible to functionally evaluate the stromal elements of the bone marrow. The whole medullary tissue is characterized by marked heterogeneity as regards its ability to synthesize humoral stimulants of granulomonocytopoiesis. The colony-stimulating ability (CSA) of this tissue combines the CSA of the hemopoietic and stromal cells. In bone marrow deficiency of varying degree, the function of the bone marrow stroma is highly activated. The trepanobiopsy areas short of the hemopoietic tissue possess a higher ability to form a monolayer on the dish surface. During AML and ALL relapses, the ability of the bone marrow stroma to synthesize the humoral stimulants is lowered, whereas the ability to form monolayers is less demonstrable. In diseases associated with the development of secondary fibrosis (CMF, CML, IP), the bone marrow stroma was discovered to be less capable of synthesizing the humoral stimulants, which provides evidence against the involvement of fibrous tissue in the formation of the total CSA of the bone marrow. It is assumed that fibroblasts and fatty cells are related histogenetically and that their proliferation is controlled by humoral factors.     

Monoclonal antibodies to Pgp-1/CD44 block lympho-hemopoiesis in long- term bone marrow cultures

A new panel of mAbs was prepared to a stromal cell line known to support lymphocytes in Whitlock-Witte type long-term bone marrow cultures. These antibodies were then screened with a cell adhesion assay and four were selected that inhibited the binding of B lineage cells to stromal cell monolayers. Immunofluorescent and biochemical analyses revealed that these new antibodies detected epitopes of the previously described Pgp-1/CD44 antigen complex. Addition of Pgp-1/CD44 antibodies to Dexter-type long-term bone marrow cultures completely prevented emergence of myeloid cells and they also blocked lymphocyte growth in Whitlock-Witte type cultures. mAbs MEL-14, LFA-1, and CD45R did not inhibit under the same conditions and there was no apparent relationship to Ig isotype. Adherent layers in treated cultures were not unusual in terms of morphology and the antibodies did not affect factor-dependent replication of lymphoid or myeloid progenitor cells. Therefore, the mechanism of inhibition may not involve direct toxicity to precursors or microenvironmental elements. Previous studies in humans and mice have implicated Pgp-1/CD44-related glycoproteins in the migration of peripheral lymphoid cells, as well as interactions of cells with the extracellular matrix. These findings suggest that they may also be critical for formation of lymphoid and myeloid cells within bone marrow.     

Differential elaboration of prostaglandin E2 by cells of the hemopoietic microenvironment in response to endotoxin

Eight daily intraperitoneal injections of endotoxin (LPS) induced hematologic abnormalities in mice like those previously observed with chronic inflammation, sterile abscess, and tumor bearing. By the ninth day, anemia, leukocytosis, hypocellularity of the bone marrow, and compensatory hemopoietic hyperplasia of the spleen had occurred. The suppressed hemopoietic recovery and impaired survival of mice with these abnormalities, after receiving an ordinarily sublethal dose of total body irradiation (600 cGy T.B.), confirmed their importance to the intact mouse and suggested that splenic hyperplasia was insufficient to compensate for a total body deficit of functional hemopoietic stem cells. Atrophy of hemopoietic tissue in the marrow with hyperplasia in the spleen implicated changes in the hemopoietic microenvironment to account for the different responses to endotoxin. Prostaglandin E2 (PGE2) serves as an important mediator of the inflammatory response and profoundly affects hemopoiesis. Previous studies had shown that low concentrations of PGE2 enhanced, and high concentrations suppressed erythropoiesis in vitro; therefore, we wondered whether stromal cells from the marrow's microenvironment produced more PGE2 in response to LPS than splenic stromal cells to explain the suppression of hemopoiesis in the marrow and its enhancement in the spleen. Indeed, synthesis of PGE2 in primary short-term cultures of adherent marrow stromal cells in response to LPS proved much greater than that observed in cultures of splenic stromal cells. Extending adherence times from 3 to 24 to 48 hours did not change the relationship. We believe that the results of our studies point to a role of PGE2 in the microenvironmental modulation of hemopoiesis in mice with activation of the inflammatory response.     

内吗啡肽-1对正常人骨髓基质细胞造血调控的研究

本研究探讨内吗啡肽-1对正常人骨髓基质细胞表面黏附分子细胞间黏附分子（ICAM-1）和血管细胞黏附分子（VCAM-1）,以及基质细胞分泌的细胞因子IL-6、TNF-α的调控作用。应用流式细胞术检测黏附分子ICAM-1和VCAM-1的表达,用ELISA法检测细胞因子IL-6、TNF-α的浓度。结果表明：加入内吗啡肽-1培养24小时后ICAM-1和VCAM-1表达与对照组比较有显著性差异（p〈0.05）,但内吗啡肽-1浓度的改变对于二者的表达无影响;IL-6、TNF-α的浓度实验各组数据与对照组比较无显著性差异（p〉0.05）。结论：内吗啡肽-1能调控正常骨髓基质表面ICAM-1和VCAM-1的表达;对于正常骨髓基质细胞分泌的细胞因子IL-6、TNF-α无促进或抑制作用。     

Hyaluronate can function as a cell adhesion molecule and CD44 participates in hyaluronate recognition

A cell adhesion model was previously used to select a series of monoclonal antibodies (mAbs), which were subsequently found to recognize CD44/Pgp-1. Interest in these reagents increased with the finding that they totally inhibited production of lymphoid or myeloid cells in long-term bone marrow cultures. Further investigation has now revealed that hyaluronate is a potential ligand for CD44 and that hyaluronate recognition accounts for the adhesion between B lineage hybridoma and stromal cells. The hybridoma cells adhered to hyaluronate-coated plastic wells as well as to monolayers of stromal cells. The adhesion in both cases was inhibited by treatment with hyaluronidases, and did not require divalent cations. Addition of exogenous hyaluronate also diminished binding of lymphoid cells to stromal cells. One of several mAbs to Pgp-1/CD44 was particularly effective at blocking these interactions. Since hyaluronate and Pgp-1/CD44 were present on both cell types, experiments were done to determine the cellular location of interacting molecules required for the adhesion process. Treatment of lymphoid cells with an anti-Pgp-1/CD44 antibody was more inhibitory than antibody treatment of the stromal cells. Conversely, hyaluronidase treatment of stromal cells reduced subsequent binding more than treatment of the lymphoid cells. Adhesive interactions that involve hyaluronate and CD44 could contribute to a number of cell recognition processes, including ones required for normal lympho-hemopoiesis.