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P W Kincade

Publications and source records attributed to P W Kincade.

At least 19 recordsLinked to original sources
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.

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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

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.

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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

Molecular signals for the production of lymphocytes in bone marrow.

Bone marrow culture and molecular cloning techniques have permitted rapid progress to be made in identifying molecules that stimulate or inhibit particular steps in blood cell formation. Two types of factors that influence B lymphocyte precursors are considered in this brief review. Cell adhesion molecules are probably needed to correctly position immature precursors within the marrow and some of the most important of these have been identified. Recent studies suggest that local or systemically derived hormones must also be added to the list of agents which can markedly influence the production of lymphocytes.

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PubMed1993

Antibody-induced activation of the hyaluronan receptor function of CD44 requires multivalent binding by antibody.

CD44 can function as a receptor for hyaluronan (HA). However, many cell lines and normal hematopoietic cells that express CD44 do not constitutively bind HA. A monoclonal antibody (mAb) specific for CD44 (IRAWB 14) has been described previously which induces CD44-mediated binding of HA rapidly (seconds to minutes) in some cell lines and in normal murine T cells. Of 16 CD44-specific mAb tested in the present study, only 3 exhibited this activity. Monovalent Fab fragments were prepared from two IgG2a antibodies that induce HA binding (IRAWB 14 and IRAWB 26) and used to determine whether multivalent binding was required for induction of HA receptor function. Fab from both antibodies had a tendency to form multivalent aggregates. After addition of iodoacetamide to prevent further aggregation, multimeric and monovalent forms were separated by gel filtration. This made it possible to compare the inducing activity of monovalent and multivalent antibody fragments of identical composition in the absence of Fc determinants. Multimeric forms were very active at inducing binding of fluorescein-conjugated HA (Fl-HA). Monovalent Fab fragments of both antibodies had 20- to 50-fold lower binding activity than intact antibody or multimer. IRAWB 26 Fab monomers were completely inactive in the induction of HA-binding. The observed weak inducing activity of IRAWB 14 Fab monomer could be attributed to very low levels of contaminating multimer. Induction of HA binding could also be achieved by using anti-immunoglobulin to cross-link Fab monomers of IRAWB 26. Thus, multivalent binding was required for the activation of HA binding by CD44-specific antibody, suggesting that the distribution of CD44 molecules on the cell surface is important for HA receptor function. In kinetic studies, induction of HA receptor function occurred simultaneously with antibody binding at 0 degrees C (ice water bath). Furthermore, antibody could induce HA binding in paraformaldehyde-fixed cells, which were permeable to propidium iodide and trypan blue, suggesting that intracellular signaling mechanisms were not involved in induction of receptor function. We conclude, therefore, that these CD44-specific antibodies are inducing HA binding by directly influencing the distribution of CD44 on the cell surface. The possibility of a concurrent change in CD44 conformation is not ruled out. We discuss possible mechanisms by which CD44 might be activated to bind HA in vivo.

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PubMed1993

The CD44 expressed on the earliest intrathymic precursor population functions as a thymus homing molecule but does not bind to hyaluronate.

A minute population of cells representing the earliest lymphoid-restricted precursor cells in the adult mouse thymus has been isolated recently in our laboratory. This population expresses low levels of CD4, very high levels of CD44 and has a surface antigenic phenotype similar to that of bone marrow hemopoietic stem cells. To examine the role of CD44 on these cells, and to ascertain its ligand, we analysed the ability of the early thymic precursor cells to bind hyaluronate (HA) which functions as a cell adhesion molecule and a ligand for CD44. We also examined if anti-CD44 antibodies (clones IM7.8.1 and KM201) can block the thymic precursor activity. The majority of the thymic precursor cells did not show specific HA binding, indicating that HA is not the ligand for the CD44 molecules expressed on these precursor cells. HA and CD44 interactions are therefore unlikely to play a role in development of cells at this early intrathymic precursor stage. When the purified intrathymic precursor cells were incubated with anti-CD44 antibodies before intravenous transfer into recipients, very few progeny cells were detected in the thymus, although progeny were found in the spleen and lymph nodes. Coating the cells with other isotype-matched antibodies did not have this effect. However, when the same anti-CD44-coated cells were transferred directly into the recipient thymus, normal levels of progeny cells were detected in this organ. This suggests that the CD44 on the intrathymic precursor cells is a homing molecule, able to direct cells to the thymus but utilizing some ligand other than HA.

Animals↗
PubMed1993

Analysis of inflammatory endothelial changes, including VCAM-1 expression, in murine cardiac grafts.

We have employed a murine model of cardiac transplantation and two monoclonal antibodies, M/K-2 and MECA-32, to study the responses of graft endothelia during allograft rejection. Using immunohistologic techniques, we demonstrate that the monoclonal antibody M/K-2, which binds to the murine cellular adhesion molecule VCAM-1, reacts with an inducible endothelial epitope found in rejecting cardiac allografts, but not in cardiac isografts, normal cardiac tissues, or extracardiac vasculature from allografted mice. Similar, but focal, M/K-2 reactivity is also found in nontransplanted hearts undergoing virally induced myocarditis. M/K-2 reactivity does not develop in the nonrejecting cardiac allografts from nu/nu mice, and M/K-2 reactivity is found only in grafts that develop CD25+ graft-infiltrating cells--i.e., allografts but not isografts. PCR analyses of grafts during development of VCAM-1 expression indicate that allografts, but not isografts, contain mRNA for the cytokines IL-2 and IFN-gamma, and either of these cytokines may be associated with the expression of M/K-2 reactivity in rejecting allografts. Unlike M/K-2, MECA-32 identifies an inducible epitope that is observed on myocardial endothelia of both isografts and allografts, but not normal cardiac tissues. Further, expression of the MECA-32 epitope can occur in grafts that do not develop CD25+ infiltrating lymphocytes, since it is observed in isografts and the native hearts of transplanted or sham-operated mice. Indeed, MECA-32 reactivity may be T cell independent, since it is also found in nonrejecting allografts of nu/nu mice. PCR analyses of grafts during development of MECA-32 reactivity indicate that cardiac isografts contain mRNA for IL-1, IL-6, TNF, and lymphotoxin. One or more of these might be associated with induction of MECA-32 reactivity.

Animals↗
PubMed1993

Treatment with anti-vascular cell adhesion molecule 1 monoclonal antibody induces long-term murine cardiac allograft acceptance.

Daily in vivo treatment of murine H-2d --> H-2b cardiac allograft recipients with 400 micrograms/day i.p. of M/K-2, a mAb to the endothelial adhesion molecule VCAM-1, resulted in prolongation of graft survival. The surviving allografts showed little of the histologic changes observed in acutely rejecting control allografts. When antibody treatment was discontinued after 20 days, grafts continued to function for at least 40 more days. This was approximately 30 days after mAb was no longer detectable by ELISA in the circulation, or by immunoperoxidase staining at the graft site. The most notable feature of grafts that survived 60 days was the presence of mild interstitial fibrosis. Endothelial reactivity was minimal with the mAbs MECA-32 and M/K-2, which have been used in previous studies to visualize the extensive endothelial inflammation that develops during untreated acute rejection. There was a mild cellular infiltrate containing T cells, but few macrophages. However, infiltrating T cells appeared to be inactive in that IL-2R+ cells were immunohistologically undetectable and mRNA for IL-2, IL-4, or IFN-gamma was undetectable by polymerase chain reaction. In general, the immunologic conditions in these long-term grafts differed from those seen in normal cardiac tissue, cardiac isografts, or cardiac allografts. These data demonstrate that M/K-2 mAb can suppress cardiac allograft rejection and induce long-term graft acceptance. This graft survival appears to be associated with the development of a unique state of immunity at the graft site.

Animals↗
PubMed1992

Highly restricted expression of a stromal cell determinant in mouse bone marrow in vivo.

B lymphocyte precursor cells in mouse bone marrow develop in close association with stromal cells which provide essential growth signals. To identify molecules that may normally play a role in this interaction we have examined the in vivo binding of a new monoclonal antibody (mAb) (KMI6) that recognizes a determinant on a bone marrow stromal cell line (BMS2) in vitro. Flow cytometric and radioautographic evaluations revealed that the antigen recognized by KMI6 is represented on the surface of an extremely small number of cells in bone marrow cell suspensions from adult mice. An apparent molecular mass of 110 kD was obtained by surface labeling of a stromal cell clone and immunoprecipitation. Purified mAb KMI6 labeled with 125I was then given intravenously to young C3H/HeJ mice. Unbound mAb was washed out by cardiac perfusion and femoral bone marrow was examined by light and electron microscope radioautography. KMI6 labeling was heavy on the plasma membrane of many stromal cells, especially those located towards the outer subosteal region. The KMI6-labeled stromal cells were usually associated with cells of lymphoid morphology which they often completely surrounded. The labeling was restricted to areas of stromal cell plasma membranes in contact with lymphoid cells. The lymphoid cells themselves, as well as macrophages and other hemopoietic cells, failed to bind mAb KMI6 significantly. Stromal cells in bone marrow depleted of hemopoietic cells by gamma-irradiation (9,5 Gy) bound mAb KMI6 at reduced intensity. The results demonstrate that the KMI6 determinant, a 110-kD protein, is expressed on bone marrow stromal cells in vivo. Its restriction to areas of interaction with lymphoid cells suggests a role in forming microenvironmental niches of B lymphopoiesis. The surface membrane of individual stromal cells may thus be functionally polarized towards interacting B cell precursors and other hemopoietic cells.

Animals↗
PubMed1992

B29 gene products complex with immunoglobulins on B lymphocytes.

B29 is a B-lineage-specific gene predicted from sequence information to be a transmembrane member of the immunoglobulin (Ig) superfamily, with a single extracellular Ig-like domain. Its presumptive cytoplasmic region contains a peptide motif present in CD3 and other molecules involved in lymphocyte activation. Affinity-purified goat antibodies were prepared to a TrpE fusion protein of B29 and used to study B29 expression on lymphoid cells. The antiserum precipitated surface-labeled heterodimers from B lymphoma cells. One was 65-88 kDa (unreduced) or 36-47 plus 32-34 kDa (reduced) by SDS/PAGE analysis, regardless of detergent. A smaller heterodimer was detected only with Triton detergent extraction. IgM molecules were coprecipitated by the B29 antiserum when the weak detergent digitonin was used. In addition, cocapping experiments revealed that most B29 molecules codistribute with Ig on the cell surface. Although early B-lineage cells and plasma cells contain B29 mRNA, surface expression was detectable only on B cells that had significant amounts of surface Ig. The surface expression was B-lineage-specific and included cells from mutant xid mice and B-cell lines representing mu, delta, gamma, and alpha heavy-chain isotypes and both kappa and lambda light-chain types. The density of surface B29 protein correlated directly with surface mu heavy-chain density on subclones of a B-cell lymphoma and lipopolysaccharide-stimulated pre-B cells. These findings show that B29 is covalently linked in a heterodimer and are consistent with a recently proposed model of surface Ig complexes.

Animals↗
PubMed1992

Requirements for hyaluronic acid binding by CD44: a role for the cytoplasmic domain and activation by antibody.

The CD44-negative T lymphoma AKR1 (CD44.2 genotype) was transfected with a CD44.1 cDNA. The intact cDNA conferred on the transfected cells the ability to bind hyaluronic acid (HA) both from solution and immobilized on culture plates. It also conferred a CD44-dependent and hyaluronidase-sensitive increase in adhesion to a lymph node endothelial cell line. A mutant cDNA which codes for a CD44 molecule lacking most of the cytoplasmic domain of CD44 was also transfected into AKR1, and cell sorting was used to select transfectants expressing levels of cell surface CD44 expression comparable with the line transfected with the wild-type CD44 cDNA. The cells transfected with the mutant construct bound fluoresceinated HA from solution very poorly, but did adhere to immobilized HA, though less well than cells transfected with the wild-type construct. This result indicates that the cytoplasmic domain of CD44 is necessary for binding of HA from solution but is not required for binding to immobilized HA, although it may contribute to adhesion following ligand recognition. A monoclonal antibody (mAb), IRAWB 14, which reacts with CD44 on all CD44+ cells dramatically induced HA binding by some CD44+ cell lines that did not constitutively bind HA. The transfectant expressing a CD44 molecule with a truncated cytoplasmic domain could be induced by this antibody to bind fluoresceinated-HA from solution. Splenic T cells did not bind fluoresceinated HA constitutively. In the presence of the IRAWB 14 mAb, virtually all CD44+ splenic T cells bound HA. Induction was immediate and occurred equally well at room temperature and at 4 degrees C, indicating that the new HA-binding activity was due to preexistent CD44 molecules. These results are compatible with an antibody-induced activation of CD44 by either a conformational change in the CD44 molecule or a change in the distribution of CD44 molecules on the cell surface.

Animals↗
PubMed1992

Cell interaction molecules and cytokines which participate in B lymphopoiesis.

A molecular and cellular definition of the bone marrow microenvironment is rapidly contributing to our understanding of lymphohaemopoiesis. While lineage specific genes and their protein products are being identified, information is accumulating about mechanisms which may regulate their expression. Stimulation of B lymphocyte precursor replication, and other discrete functions, are being attributed to cytokines such as interleukin 7 (IL-7). The activity of these factors may be controlled at the level of synthesis, local concentration and interaction with extracellular matrix. Extremely small amounts of IL-7 are made by stromal cells, which are themselves being thoroughly studied as cloned cell lines. This in vitro characterization suggests that stromal cells can make at least 12 cytokines, that they can respond to some of those cytokines themselves, and that they retain differentiation potential. Several molecules have been identified which are probably required for recognition between cells in marrow. It is noteworthy that they belong to several previously described families of adhesion molecules and none is unique to that tissue. VCAM-1 is constitutively expressed on stromal cells in marrow and can be recognized by pre-B cells which bear the integrin VLA-4. The same pair of molecules is probably responsible for extravasation of leukocytes in other tissues during inflammation. Cell adhesion molecules are likely to work in a carefully coordinated and cooperative fashion. Their activity can be controlled by expression or, in some cases, modulated after display on the cell surface. For example, while most haemopoietic cells bear CD44, only certain cells utilize it for recognition of the ligand hyaluronate. The affinity for hyaluronate can be experimentally regulated and depends on the cytoplasmic domain of CD44. This capability for dynamic change may be important for transient interactions between cells, permitting movement of maturing precursors within and from marrow.

Animals↗
PubMed1992

Molecular isoforms of murine CD44 and evidence that the membrane proximal domain is not critical for hyaluronate recognition.

We previously found that the CD44 glycoprotein on some lymphocytes can mediate adhesion to hyaluronate (HA) bearing cells. However, many questions remain about the molecular heterogeneity of CD44 and mechanisms which control its recognition of this ligand. In vitro mutagenesis and DNA sequencing have now been used to investigate the importance of the membrane proximal region of murine CD44 for recognition of soluble or cell surface HA. CD44 with an 83 amino acid deletion in this region mediated binding to soluble ligand and the apparent avidity increased markedly in the presence of a particular antibody to CD44, IRAWB14. The shortened CD44 was however inefficient in mediating adhesion of transfected cells to HA immobilized on cell surfaces. Four new murine isoforms of CD44 were isolated from a carcinoma line by use of the polymerase chain reaction. Only two of them correspond to ones recently discovered in rat and human cells. The longest variant nearly doubled the length of the extracellular portion of the molecule and introduced an additional 20 potential sites for glycosylation. When expressed on T lymphoma cells, all four of the new murine CD44 isoforms were capable of mediating adhesion to HA bearing cells. This result contrasts with a report that a related human CD44 isoform lacks this ability when expressed on B lineage lymphoma cells. The new murine isoforms also conferred the ability to recognize soluble HA and were very responsive to the IRAWB14 antibody. A brief survey of normal murine cell lines and tissues revealed that the hemopoietic isoform was the most abundant species. These findings indicate that the NH2-terminal portion of CD44 is sufficient for HA recognition and that this function is not necessarily abrogated by variations which occur in the membrane proximal domain. They add to the known molecular diversity of CD44 and provide another experimental model in which isoform specific functions can be investigated.

Amino Acid Sequence↗
PubMed1991

Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis.

Adhesion molecules are probably required for retention of maturing lymphocyte precursors in bone marrow, where they closely interact with and are dependent on stromal cells. Lymphomyeloid cell lines avidly adhere to cloned stromal cell lines in culture and screening pairs of these resulted in a selection strategy for a new monoclonal antibody to a leukocyte adhesion molecule. Immunoprecipitation analyses and comparison to a previously described antibody showed that it recognizes the alpha 4 chain of the integrin, VLA-4. This antibody totally inhibited lymphopoiesis and retarded myelopoiesis in long-term bone marrow cultures. A similar selection strategy resulted in two additional antibodies which define a single 100-kD species on stromal cells. This stromal cell adhesion molecule is a potential counter-receptor/ligand for VLA-4 on murine lympho-myeloid cells. Our findings suggest a new role for VLA-4 in lymphoid progenitor-microenvironment interactions. Recognition molecules that function in cell migration and inflammation in peripheral tissues may be important for steady-state lymphopoiesis within bone marrow.

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