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P W Kincade

Publications and source records attributed to P W Kincade.

At least 19 recordsLinked to original sources
PubMed1997

Indirect suppression of IL-7-responsive B cell precursors by vasoactive intestinal peptide.

Bone marrow is supplied with nerves and neuropeptides that influence a variety of cellular responses. This study represents an initial evaluation of vasoactive intestinal peptide (VIP) as a possible regulator of B lineage lymphocyte formation. As little as 10(-10) M concentrations of VIP inhibited the IL-7-driven clonal proliferation of pre-B cells in semisolid agar cultures. The response was blocked by a VIP antagonist and augmented by the ectoenzyme inhibitor, phosphoramidon. Suspensions of highly enriched B lineage precursors were unaffected by VIP unless they were cocultured with macrophage-like cells and conditioned medium from VIP-treated macrophages contained inhibitory activity. Neutralizing Abs were used to determine that IFN-alpha is at least one substance that is elicited by exposure of macrophages to VIP. These findings suggest that a neuropeptide can potentially modulate lymphopoiesis through a regulatory circuit that involves macrophages and IFN-alpha. They also raise the possibility that VIP can participate in antiviral defense.

Animals↗
PubMed1997

CD44-stimulated dendrite formation ('spreading') in activated B cells.

A rat monoclonal antibody (mAb) (NIM-R8), insolubilized by binding to plastic plates, induced a rapid and extensive formation of dendrite processes ('spreading') in B lymphocytes activated by anti-IgM and interleukin-4 (IL-4) or anti-CD38 and IL-4. In contrast, resting B cells were unable to spread similarly on the NIM-R8-coated plates. The NIM-R8 antibody recognized a 90,000 MW surface glycoprotein (gp90) present on both B and T lymphocytes. The expression of this molecule was greatly increased after polyclonal (lipopolysaccharide, anti-IgM plus IL-4 or concanavalin A) activation. The NIM-R8 mAb with or without IL-2 or IL-4 was unable to induce proliferation of splenic lymphocytes. Following the demonstration that the NIM-R8 mAb recognizes the murine equivalent of human CD44, the induction of spreading of activated B lymphocytes was studied using a panel of mAb recognizing different epitopes of murine CD44. All of these different mAb induced similar spreading of activated B cells. The ligand-inducible spreading of activated B lymphocytes may be an important mechanism for providing an increased cell-surface area for cell-cell or cell-matrix interactions, and thus may be an important factor controlling the response of activated lymphocytes.

Animals↗
PubMed1996

Identification and functional analysis of Ly-6A/E as a thymic and bone marrow stromal antigen.

We recently described an mAb (MTS23) reactive with a membrane Ag expressed on a subset of thymic medullary stromal cells. This Ag is also constitutively expressed at high levels on peripheral B cells, macrophages, and thymic and splenic dendritic cells of C57BL/6 mice. A number of stromal cell lines derived from thymus and bone marrow also stain with MTS23, but thymocytes and peripheral T cells only weakly express the Ag detected by MTS23. Here we show that the molecule detected by MTS23 is a member of the Ly-6 family of phosphatidylinositol-anchored membrane proteins. Treatment of stromal cells with phosphatidylinositol-phospholipase C before staining completely abolished expression. Using transient expression of 293T cells and a cDNA library of a stromal cell line cloned into the pEF-BOS vector, a cDNA encoding the MTS23-target Ag was isolated. Partial sequencing and restriction enzyme mapping revealed that it represents the Ly-6A/E protein. While the physiologic significance of the presence of Ly-6 molecules on stromal cells is not clear, it has been known for some time that, at least in lymphocytes, cellular activation events can be induced upon Ly-6 engagement. We now demonstrate that Ly-6 also functions as a signal transduction molecule on stromal cells, in that granulocyte-macrophage CSF can be produced by a variety of stromal cell lines upon mAb-mediated cross-linking of Ly-6. Together with the dramatic up-regulation of Ly-6 expression on stromal cells upon IFN-gamma treatment, this is the first indication of a biologic function of an Ly-6 gene product on nonhemopoietic cells. The results suggest that Ly-6 may play a role in the cross-talk between lymphocyte precursors and stromal cells.

Animals↗
PubMed1996

Antibody ligation of CD9 modifies production of myeloid cells in long-term cultures.

The KMC8.8 monoclonal antibody was made by immunizing rats with the BMS2 stromal cell clone, and was selected for further study because its ability to inhibit production of myeloid cells in Dexter cultures but not that of lymphoid cells in Whitlock-Witte cultures. The influence on myeloid progenitors might have been indirect, since the antibody did not prevent responsiveness to colony-stimulating factors in semisolid agar cultures. Furthermore, there was no inhibition, and some augmentation, of cell production when the antibody was added to established Dexter cultures. A cDNA clone that encoded the KMC8.8-recognized molecule was isolated by expression cloning and found to be identical in sequence to a previously published murine CD9 homologue. The antibody and cDNA clone were used to establish that CD9 is expressed by stromal cells, megakaryocytes, platelets, myeloid cells, and subpopulations of mature lymphocytes in mice. Treatment with the KMC8.8/CD9 antibody slightly augmented adhesion between myeloid cells and stromal cells, consistent with previous reports that this member of the tetraspan family of proteins can transmit proadhesive signals to human platelets and lymphoid cells. CD9 might participate in cell-cell interactions critical for correct orientation and movement of maturing myeloid cells in bone marrow.

Animals↗
PubMed1996

Adhesion receptors on bone marrow stromal cells: in vivo expression of vascular cell adhesion molecule-1 by reticular cells and sinusoidal endothelium in normal and gamma-irradiated mice.

Cell adhesion molecules (CAMs) play a key role in interactions between stromal and hematopoietic cells in bone marrow (BM) and in cell traffic through vascular endothelium. To examine the identity of CAMs involved in these processes in mouse BM, we have investigated the in vivo expression of vascular cell adhesion molecule-1 (VCAM-1) and its counter-receptor, very late antigen-4 (VLA-4). Radioiodinated monoclonal antibodies (MoAbs) detecting VLA-4 and VCAM-1 were injected intravenously. Antibody binding was detected in BM by light and electron microscope radioautography. VCAM-1 labeling was restricted to stromal reticular cells and endothelial cells lining BM sinusoids. VCAM-1+ reticular cells formed patchy concentrations, especially in subosteal regions, associated with lymphoid, granulocytic, and erythroid cells. After gamma-irradiation to deplete hematopoietic cells, reticular cells and endothelial cells all showed VCAM-1 labeling in apparently increased intensity. VLA-4 labeling was shown by undifferentiated blast cells and lymphohematopoietic cells both in BM cell suspensions and in vivo, especially at reticular cell contact points. The results demonstrate that VCAM-1 is expressed in vivo by certain BM reticular cells, suggesting that the molecule mediates adhesion to multiple lineages of lymphohematopoietic cells. The finding that VCAM-1 is also expressed constitutively by BM sinusoidal endothelium, unlike its inductive expression by endothelia elsewhere, suggests that VCAM-1 and VLA-4 may be involved in regulating the normal cell traffic between BM and the blood stream.

Anemia, Aplastic↗
PubMed1996

Identification of stromal cell products that interact with pre-B cells.

Our understanding of lympho-hematopoietic microenvironments is incomplete, and a new cloning strategy was developed to identify molecules that bind to B lineage lymphocyte precursors. A cell sorting procedure was used for initial enrichment of cDNAs from stromal cell mRNA that contained signal sequences and were therefore likely to encode transmembrane or secreted proteins. A second step involved expression of the library as soluble Ig fusion proteins. Finally, pools representing these proteins were screened for the ability to recognize pre-B cells. This approach resulted in the cloning of biglycan, syndecan 4, collagen type I, clusterin, matrix glycoprotein sc1, osteonectin, and one unknown molecule (designated SIM). The full-length cDNA of SIM revealed that it is a type I transmembrane protein, and its intracellular domain has weak homology with myosin heavy chain and related proteins. Staining of established cell lines and freshly isolated hematopoietic cells with the Ig fusion proteins revealed distinct patterns of reactivity and differential dependence on divalent cations. Biglycan-, sc1-, and SIM-Ig fusion proteins selectively increased interleukin 7-dependent proliferation of pre-B cells. Overexpression of the entire SIM protein affected the morphology of 293T cells, while expression of just the extracellular portion was without effect. Thus, a series of stromal cell surface molecules has been identified that interact with blood cell precursors. Three of them promoted the survival and/or proliferation of pre-B cells in culture, and all merit further study in relation to lympho-hematopoiesis.

Amino Acid Sequence↗
PubMed1995

Estrogen suppresses stromal cell-dependent lymphopoiesis in culture.

Numbers of pre-B cells change dramatically and reciprocally in response to estrogen levels in mice, suggesting that normal lymphopoiesis may be under hormonal control. However, little is known of the mechanisms involved in this process. We found that estrogen receptor mRNA was detectable by RT-PCR in lymphocyte supporting stromal cells as well as B lymphocyte precursors. Unlike glucocorticoids, estrogen did not induce apoptosis in isolated B lineage lymphocytes or interfere with their responsiveness to IL-7 in semisolid agar. Estrogen did inhibit clonal expansion of B cell precursors in a limiting dilution-type assay when the lymphocytes were cultured on a stromal cell clone. In other experiments, B cell precursors at particular stages of differentiation were isolated by cell sorting and cocultured with stromal cells for 4 days. This revealed that some subsets were more sensitive to an estrogen-containing environment than others. Although numbers of recovered cells were greatly reduced, the remaining lymphocytes had undergone relatively normal differentiation. The surviving population was enriched in cells that had acquired cytoplasmic mu chains, BP-1 Ag, and clonability with IL-7. Hormone-mediated inhibition occurred in serum and phenol-red free medium, and in cultures replete with IL-7. Direct contact between stromal cells and lymphocytes was not required. Furthermore, suppression resulted when stromal cells alone were treated with the hormone. These findings indicate that estrogen may regulate B lymphopoiesis via its influence on the microenvironment and that estrogen-induced stromal cell genes merit further study.

Animals↗
PubMed1995

Glycosylation of CD44 negatively regulates its recognition of hyaluronan.

Although CD44 is expressed on a wide variety of cell types, few of them use it to recognize the ligand hyaluronan (HA). A glycosylation-defective clone of Chinese hamster ovary cells (Lec 8) bound HA, demonstrating that complete processing of glycoproteins with addition of a full complement of sialic acid is not required. On the contrary, subsequent findings revealed that complex sugars on CD44 can actually inhibit ligand recognition. Two subclones of wild-type Chinese hamster ovary cells with similar amounts of surface CD44 were isolated on the basis of HA binding and found to differ with respect to CD44 size as well as staining with fluorescent lectins. Treatment of the nonbinding clone with tunicamycin reduced the size of the protein and allowed the cells to recognize HA via CD44. This function was also induced by treatment with deglycosylating enzymes (either a mixture of endoglycosidase F and N-glycosidase F or neuraminidase alone). A possible role for glycosylation in regulation of adhesion was then sought with a series of normal and transformed murine cells. Disruption of glycosylation or treatment with deglycosylating enzymes did not induce ligand binding in an interleukin 7-dependent pre-B cell line, and splenic B cells also appeared to be in an inactive state. Some normal B cells acquired the ability to recognize HA after stimulation with lipopolysaccharide or interleukin 5 and had distinctive surface characteristics (loss of immunoglobulin D and acquisition of CD43). An additional subset of activated cells might have been in a transitional state, because the cells bound ligand after neuraminidase treatment. The ligand-binding ability of a purified CD44-immunoglobulin fusion protein dramatically increased after neuraminidase treatment. Thus, differential glycosylation of this molecule is sufficient to influence its recognition function. Cell adhesion involving HA can be regulated by multiple mechanisms, one of which involves variable glycosylation of CD44.

Animals↗
PubMed1995

Monoclonal antibodies to CD44 and their influence on hyaluronan recognition.

Antibodies to CD44 have been used to inhibit a variety of processes which include lymphohemopoiesis, lymphocyte migration, and tumor metastasis. Some, but not all, CD44-mediated functions derive from its ability to serve as a receptor for hyaluronan (HA). However, sites on CD44 that interact with either ligands or antibodies are poorly understood. Interspecies rat/mouse CD44 chimeras were used to analyze the specificity of 25 mAbs and to determine that they recognize at least seven epitopes. Amino acid substitutions that resulted in loss of antibody recognition were all located in the region of homology to other cartilage link family proteins. While at least five epitopes were eliminated by single amino acid replacements, multiple residues had to be changed to destroy binding by other antibodies. One antibody was sensitive to changes in any of three separate parts of the molecule and some antibodies to distinct epitopes cross-blocked each other. Certain antibodies had the ability to increase HA binding by lymphocytes but this did not correlate absolutely with antibody specificity and was only partially attributable to CD44 cross-linking. Antibodies that consistently blocked HA recognition were all sensitive to amino acid changes within a short stretch of CD44. Such blocking antibodies interacted with CD44 more strongly than ligand in competition experiments. One large group of antibodies blocked ligand binding, but only with a particular cell line. This detailed analysis adds to our understanding of functional domains within CD44 and requirements for antibodies to influence recognition of one ligand.

Amino Acid Sequence↗
PubMed1994

Characterization of soluble CD44 in the circulation of mice. Levels are affected by immune activity and tumor growth.

ELISA determinations revealed substantial concentrations (0.49 to 2.10 micrograms/ml) of soluble CD44 in murine serum, with some variation among normal mouse strains. At least three species of CD44 were identified by immunoprecipitation and SDS-PAGE analysis of serum. The most prominent was indistinguishable in mobility from that extracted from normal and transformed lymphocytes and was estimated in this way to be approximately 90 kDa. A similar estimate resulted from gel filtration under nondenaturing conditions, followed by ELISA. However, lymphocyte membrane-extracted and soluble CD44 had different mobilities after treatment with neuraminidase plus O-glycosidase, and the core protein of soluble CD44 might be 17 to 20 kDa smaller than that of CD44 on lymphocyte membranes. Furthermore, an Ab to cytoplasmic residues of CD44 failed to recognize soluble CD44 recovered from the circulation or in lymphoma culture supernatants. These observations would be consistent with cleavage of CD44 from cell surfaces; and protease inhibitors slowed the loss of CD44 from cultured lymphomas. Serum CD44 levels were significantly reduced in immunodeficient CD17.SCID and BALB/c.Xid mice, and elevated in tumor-bearing mice. Mild graft-vs-host (GVH) reactions also resulted in increased concentrations of CD44, as did autoimmune disease in BXSB and MRL/lpr strains of mice. Serum with high concentrations of CD44 partially blocked the binding of one ligand, hyaluronate, to CD44-bearing hybridoma cells. The degree of inhibition was positively correlated with CD44 concentration. These findings indicate that substantial quantities of CD44 can be released into the circulation by cleavage from cell surfaces and that this process is markedly influenced by immune system activity and tumor growth. The material seemed to be intact and potentially functional.

Amino Acid Sequence↗
PubMed1994

Increased B lymphopoiesis in genetically sex steroid-deficient hypogonadal (hpg) mice.

Interleukin 7 (IL-7) responsive B lineage precursors were greatly expanded in genetically hypogonadal female (HPG/Bm-hpg/hpg) mice that have a secondary deficiency in gonadal steroidogenesis. Estrogen replacement in these mice resulted in a dose-dependent reduction in B cell precursors. More modest increases were documented in genetically normal mice that were surgically castrated. These findings complement other recent observations that B lymphopoiesis selectively declines in pregnant or estrogen-treated animals. Sex steroids have long been known to influence such disparate processes as bone physiology and tumor growth, in addition to their importance for reproductive function. We now show that these hormones are important negative regulators of B lymphopoiesis.

Animals↗
PubMed1994

Pregnancy-related steroids are potential negative regulators of B lymphopoiesis.

B lymphopoiesis is selectively suppressed in normal pregnant mice, suggesting that fluctuations in systemic hormone levels might influence local events within bone marrow. This has now been tested by sustained experimental elevation of sex steroids by hormone-containing pellet implants. We found that while numbers of total nucleated cells declined after treatment with estrone, beta-estradiol, or estriol, there was preferential suppression of B-lymphocyte lineage precursors. Progesterone pellets had no effect when used alone, but mice exposed to progesterone were sensitive to several-logarithm lower concentrations of estrogen. Changes in subpopulations of B-lymphocyte lineage cells with hormone pellets were similar to those previously recorded in pregnancy. B-lymphocyte lineage precursors in male and female mice were sensitive to these sex hormones. Acute treatment with single injections of water-soluble beta-estradiol allowed temporal effects on B-lineage cells to be documented. With this protocol, total numbers of nucleated cells and myeloid progenitor cells remained unchanged. Interleukin 7-responsive precursors dramatically declined within 1 day of injection, suggesting that estrogen influences that stage in the B-lymphocyte lineage. There was a subsequent sharp drop in small pre-B cells 4 days after this transient elevation in estrogen. These experiments demonstrate that B lymphopoiesis is sensitive to negative regulation by sex steroids. They extend findings made with pregnant animals and parallel previous studies of the thymus. Sex steroids might contribute to control of steady-state lymphopoiesis, and fluctuations in their levels could have implications for human disease.

Animals↗
PubMed1994

Native associations of early hematopoietic stem cells and stromal cells isolated in bone marrow cell aggregates.

In suspensions of murine bone marrow, many stromal cells are tightly entwined with hematopoietic cells. These cellular aggregations appear to exist normally within the marrow. Previous studies showed that lymphocytes and stem cells adhered to stromal cells via vascular cell adhesion molecule 1 (VCAM1). Injection of anti-VCAM1 antibody into mice disrupts the aggregates, showing the importance of VCAM1 in the adhesion between stromal cells and hematopoietic cells in vivo. Early hematopoietic stem cells were shown to be enriched in aggregates by using a limiting-dilution culture assay. Myeloid progenitors responsive to WEHI-3CM in combination with stem cell factor (c-kit ligand) and B220- B-cell progenitors responsive to insulin-like growth factor-1 in combination with interleukin-7 are not enriched. We propose a scheme of stromal cell-hematopoietic cell interactions based on the cell types selectively retained within the aggregates. The existence of these aggregates as native elements of bone marrow organization presents a novel means to study in vivo stem cell-stromal cell interaction.

Animals↗
PubMed1994

Pregnancy: a clue to normal regulation of B lymphopoiesis.

The large-scale production of lymphocytes in the bone marrow reflects a delicate balance between positive and negative regulatory signals. For instance, interleukin 7 (IL-7) provides a positive signal, and appears to be both essential and limiting in the mouse. However, much less is understood concerning the negative molecular signals that may limit the output of lymphocytes. Here, Paul Kincade and colleagues discuss how a chance observation with pregnant mice revealed that sex steroids can act as negative regulators of B lymphopoiesis, and may do so under normal steady-state conditions.

Animals↗
PubMed1994

B-lineage differentiation in normal and transformed cells and the microenvironment that supports it.

B-cell differentiation is a complex process mediated through interactions with the microenvironment of the bone marrow and fetal liver. These interactions alter patterns of gene expression and allow precursors to develop into Ig+ B cells. Recent work has shown that some of these events can be triggered in B-cell precursors transformed by Abelson virus. Other advances have refined our understanding of the role of cytokines, hormones and stromal cells in the differentiation process.

Abelson murine leukemia virus↗
PubMed1994

Recognition of murine integrin beta 1 by a rat anti-stromal cell monoclonal antibody.

Previous studies with the rat monoclonal antibody KMI6 had localized its antigen in vivo to a discrete subpopulation of marrow stromal cells. The KMI6 antigen has now been identified as the murine homolog of integrin beta 1 by amino acid sequence analysis and by cross-reactivity with antiserum to the avian integrin beta 1. The relative tissue abundance of murine integrin beta 1 was determined by Western blot. Although immunoperoxidase staining of fixed murine hematopoietic tissues demonstrated an abundance of intracellular beta 1, few primary-derived cells of lymphohematopoietic origin were surface positive as assessed by immunofluorescence and flow cytometry. Fetal erythroblasts provided the only exception. In contrast, the antigen was readily detected on the surface of several cultured cell lines in association with a variety of alpha chains. The biochemical properties of the surface labeled murine integrin beta 1 were similar to those of its human counterpart, exhibiting an altered electrophoretic migration under reduced conditions or following N-glycanase treatment. The antibody recognition of the protein was insensitive to glycosylation state, presence of divalent cations, detergents, or transfer to a nitrocellulose membrane. However, on Western blot, the epitope was lost on reduction of the protein, suggesting that it is conformation dependent. These data indicate that although KMI6 epitope is widely distributed, its surface expression in vivo may be restricted within lymphohemopoietic tissues.

Amino Acid Sequence↗
PubMed1994

Sex hormones as negative regulators of lymphopoiesis.

B lymphocytes, together with cells of seven other lineages, are made in large numbers from precursors in the bone marrow. Using cell culture models and recombinant proteins, progress has been rapid in identifying cytokines which could potentially regulate the proliferation, differentiation and migration of B-cell precursors. However, we still know little about molecular mechanisms which are important for maintaining steady-state conditions in vivo. B lymphopoiesis is severely diminished during pregnancy in normal mice and this provided a clue that sex hormones might be important negative regulators. Administration of estrogens alone, or in combination with progesterone, preferentially suppressed IL-7 responding cells and their progeny in bone marrow. There is precedent for these observations in the thymus, which transiently involutes during pregnancy, and also atrophies following estrogen treatment. The actual mechanism(s) through which sex steroids influence lymphopoiesis remain unclear, but cell culture experiments should be informative about potential interactions between hormones, the bone marrow microenvironment, and lymphocyte precursors. These findings raise a number of other important issues. For example, we need to learn if sex steroids are produced and/or concentrated locally within the marrow, if human lymphopoiesis is sensitive to these hormones, and if production of lymphocytes can be augmented in aging and in immunodeficiency by hormone manipulation.

Animals↗