The malignant process is, in many aspects, a distorted image of normal physiological activities. In this respect, the dissemination mechanism of malignant lymphomas may display a hazy reflection of normal cell migration, an essential function of the lymphoid system. The successful response of the individual’s defense machinery against invading microorganisms is largely due to its ability to rapidly mobilize leukocytes to the site of infection. Cell motility in blood, lymph, lymphoid organs and tissues is highly dependent on the coordinated activity of different cell adhesion molecules. These are implicated in the transendothelial migration of intravasated and extravasated cells, the capture of cells by the luminal surface of the endothelium, cell rolling and cell arrest in the vasculature, binding of lymphocytes to the high endothelial venule (HEV) of the lymph node, as well as subsequent cell lodgment in organ parenchyma (a process known as cell homing), and cell migration on extracellular matrix (ECM) (Parkhurst and Saltzman 1992; Picker and Butcher 1992; Thomas et al. 1992; Springer 1994; Ley and Tedder 1995). Three pairs of adhesion receptor and counterreceptor families are implicated in the interaction between leukocytes and their target cells in the endothelium and tissues: (1) integrins, which target molecules of the immunoglobulin superfamily (IgSF) or ECM components, (2) selectins, which interact with sialylated carbohydrate determinants O-linked to mucin-like molecules (also known as addressins), and (3) CD44 receptors with binding affinity for matrix and cell surface constituents (Ruoslahti 1991; Yamada 1991; Picker and Butcher 1992; Lesley et al. 1993; Springer 1994; Naor et al. 1997).

To analyze the role of alpha4-integrins in lymphoma metastasis, sublines of the T-cell lymphoma LB were generated by retrovirus-mediated gene transfer that differ exclusively in the expression of alpha4-integrins. Using LB-alpha4 and control LB-NTK cells, we demonstrate that expression of alpha4-integrins strongly suppresses metastasis formation of LB lymphoma cells in secondary lymphoid organs such as spleen, mesenteric and peripheral lymph nodes, or Peyer's patches after i.v. injection into syngeneic BALB/c mice. Moreover, alpha4-integrin expression inhibited development of metastatic tumors in liver, lung, and kidney. Expansion of LB lymphoma cells in bone marrow was not affected by alpha4-integrin expression. In vivo migration assays using 51Cr-labeled lymphoma cells demonstrated that low-metastatic LB-alpha4 cells accumulated with the same efficiency as high-metastatic LB-NTK cells in all target organs examined and were even enriched in mucosal lymphoid organs. Collectively, these results indicate that alpha4-integrins inhibit metastasis formation of lymphoma cells at a stage subsequent to the invasion of target organs.

The MAdCAM-1 adhesion molecule is involved in lymphocyte homing into mucosal sites and is expressed on high endothelial venules of Peyer's patches and mesenteric lymph nodes, In the spleen, where high endothelial venules are absent, expression can be found on cells in the marginal zone between red and white pulp. By immunohistochemistry and electron microscopy it was demonstrated that in the spleen cells expressing MAdCAM-1 belong to the population of sinus-lining cells and that the expression is restricted to the sinus-lining cells closest to the lymphoid white pulp. Lymphocytes that migrate from the blood into this white pulp area will have to pass through the rim of cells expressing MAdCAM-1. A functional role for MAdCAM-1 or its lymphocyte ligand, the alpha 4 beta 7 integrin complex, was investigated by in vivo short-term homing experiments with anti-MAdCAM-1 and anti-alpha 4 beta 7 antibodies, but no direct role for this receptor-ligand interaction could be demonstrated.

Similar to activated T cells, LB T cell lymphoma expresses the CD44 cell surface Ag. In addition, the vast majority of LB cells also express the beta 2 (CD18) and alpha L (CD11a) chains of LFA-1 integrin. In view of the finding that anti-CD18 mAb blocked spleen, but not lymph node invasion by LB cells inoculated s.c. into BALB/c mice, we tested the ability of anti-CD44 mAb (IM 7.8.1) to block the infiltration of LB cells into the lymph nodes. We found that, as opposed to anti-CD18 mAb, anti-CD44 mAb, as well as its F(ab')2 or Fab fragment, prevented lymph node infiltration but had no effect on spleen invasion. This conclusion was based on histologic examination and [3H]thymidine incorporation into proliferating LB cells invading the lymphoid organs. Histologic analysis further demonstrated that LB cells invade the lymph node via the afferent lymphatics. The surface expression of CD44 molecules on LB cells was enhanced after PMA activation. PMA activation also enabled in vitro binding of the lymphoma to hyaluronic acid (HA), a known ligand of CD44. Because anti-CD44 mAb, its F(ab')2 or Fab fragment, and hyaluronidase blocked this binding, we also tested the ability of the enzyme to inhibit lymph node invasion by LB cells. We established through histologic examination and [3H]thymidine incorporation that hyaluronidase protected the lymph node, but not the spleen, from invasion by the lymphoma.

The vascular cell adhesion molecule-1 (VCAM-1) plays an important role in diverse physiological and pathological processes. The homologous first and fourth immunoglobulin-like domains of the seven domain form of VCAM-1 present binding motifs for alpha 4 beta 1 integrin. Using a panel of VCAM-1 domain deletion mutants we show that alpha 4 beta 7 integrin interacts with both domains 1 and 4. In contrast to their identical domain usage, alpha 4 beta 1 and alpha 4 beta 7 integrins differ in the activation states required for binding to domains 1 and 4 of VCAM-1. We show that integrin alpha 4 beta 1 required significantly higher concentrations of Mn2+ than integrin alpha 4 beta 7 to support half-maximal adhesion to domain 4. Moreover, a clear difference in the capacity of integrins alpha 4 beta 1 and alpha 4 beta 7 to interact with domain 4 was detected in the presence of Ca2+ and Mg2+ cations. Adhesion to domain 1 of VCAM-1, however, was not affected by integrin heterodimer composition. Instead, the activity level of integrin alpha 4 beta 1 for domain 1 binding was regulated by CD24 expression. Binding to seven domain VCAM-1 was not altered significantly by beta 1 and beta 7 subunits or CD24. These data indicate that integrin heterodimer composition and CD24 expression differentially modulate integrin binding to domains 1 and 4 of VCAM-1. Mechanisms that alter integrin binding specificity or monovalent versus divalent interactions may affect the strength of adhesion as well as signal transmission in adherent cells and may therefore be critical to controlling the cellular response to integrin occupancy.

The vascular cell adhesion molecule-1 (VCAM-1) plays an important role in diverse physiological and pathological processes. The homologous first and fourth immunoglobulin-like domains of the seven domain form of VCAM-1 present binding motifs for α4β1 integrin. Using a panel of VCAM-1 domain deletion mutants we show that α4β7 integrin interacts with both domains 1 and 4. In contrast to their identical domain usage, α4β1 and α4β7 integrins differ in the activation states required for binding to domains 1 and 4 of VCAM-1. We show that integrin α4β1 required significantly higher concentrations of Mn than integrin α4β7 to support half-maximal adhesion to domain 4. Moreover, a clear difference in the capacity of integrins α4β1 and α4β7 to interact with domain 4 was detected in the presence of Ca and Mg cations. Adhesion to domain 1 of VCAM-1, however, was not affected by integrin heterodimer composition. Instead, the activity level of integrin α4β1 for domain 1 binding was regulated by CD24 expression. Binding to seven domain VCAM-1 was not altered significantly by β1 and β7 subunits or CD24. These data indicate that integrin heterodimer composition and CD24 expression differentially modulate integrin binding to domains 1 and 4 of VCAM-1. Mechanisms that alter integrin binding specificity or monovalent versus divalent interactions may affect the strength of adhesion as well as signal transmission in adherent cells and may therefore be critical to controlling the cellular response to integrin occupancy.

The mouse CD8(+) T cell lymphoma TK1 expresses high levels of alpha(4) beta(7) integrin, which it can use to interact with multiple ligands including mucosal addressin-1 (MAdCAM-1), VCAM-1, and fibronectin. In addition, alpha(4) beta(7) can support TK1 cell aggregation. Here we have produced and characterized a panel of mAbs against alpha(4) beta(7) to define antigenic and functional epitopes associated with its distinct functions. One mAb, DATK32, is unique in recognizing an epitope specific to the alpha(4) beta(7) heterodimer. Furthermore, DATK32 induces TK1 cell aggregation yet inhibits TK1 cell adhesion to MAdCAM-1, VCAM-1, and fibronectin. Considered as a whole, the panel of anti-alpha(4) beta(7) mAbs studied define unique patterns of inhibition for alpha(4) beta(7) binding to each of its defined molecular ligands. We conclude that alpha(4) beta(7) interactions with MAdCAM-1, VCAM-1, and fibronectin can be modulated by Ab binding to distinct epitopes and thus probably involve functionally separable, although physically overlapping binding sites on this multifunctional integrin. These findings are consistent with the general observation that integrins use distinct, potentially differentially regulated interaction sites for adhesion to multiple ligands. Extension of these concepts to alpha(4) beta(7) has important considerations for understanding the roles of this integrin in lymphocyte homing to mucosal sites and in cell-cell interactions during the immune response.

Lymphocyte recirculation through different organs is thought to be regulated by adhesion molecules ("homing receptors") recognizing tissue-specific vascular addressins on endothelium. Here we show that the alpha 4/beta 7-integrin has a key role in the migration of mouse lymphocytes to mucosal sites. Homing to Peyer's patches but not to peripheral lymph nodes is inhibited by Fab fragments of mAb PS/2 against the alpha 4-integrin chain, by mAb DATK32 recognizing a combinatorial epitope on the alpha 4/beta 7-integrin, and by mAb FIB30 against the beta 7-chain. The Abs significantly reduce homing of lymphocytes to the intestine, as well. The migration of immunoblasts to gut and gut-associated lymphoid tissue also involves the alpha 4/beta 7-integrin heterodimer. Another anti-alpha 4 Ab, R1-2, which blocks lymphocyte binding to Peyer's patches in the Stamper-Woodruff frozen section assay and lymphocyte adhesion to VCAM-1 and fibronectin, has only minor effects on lymphocyte traffic in vivo. Anti-VCAM-1 Ab as well as the fibronectin peptide CS-1 are without influence on the migration to Peyer's patches or intestine, in contrast to Ab against the mucosal addressin MAdCAM-1. Thus, homing to gut-associated sites is regulated by the alpha 4/beta 7-integrin heterodimer interacting with the vascular addressin, MAdCAM-1, and not with fibronectin or VCAM-1 as counterstructures. Inhibition of homing to Peyer's patches and intestine by the anti-integrin Abs studied was only partial. L-selectin also participates in the homing of small lymphocytes to mucosal sites, especially Peyer's patches, but does not contribute substantially to the localization of blasts into the intestinal wall. The results support a major, but not exclusive role of the alpha 4/beta 7-integrin in lymphocyte traffic to mucosal sites.

The immune response to foreign antigens takes place in lymphoid organs. Lymphocytes are largely mobile cells recirculating from the blood into and out of lymphoid organs (Gowans and Knight 1964; Howard etal. 1972; Gutman and Weissman 1973). Each lymphocyte is clonally predetermined to recognize a very restricted antigenic shape, and therefore lymphoid organs are required to bring together rare cells (in terms of antigen specificity) that must interact with each other. The recirculation of lymphocytes throughout the body plays a critical role in the normal function of the immune system by maximizing interactions of lymphocytes with antigen and by increasing collaborative interactions between many disparate cell types. Organ-specific lymphocyte homing is also important in augmenting the immune response in a tissue by enhancing circulation of the immunocompetent lymphocytes, mostly in the type of tissue where they first encountered antigen. The specific portal of entry of lymphocytes from the blood stream into lymphoid organs was identified as specialized postcapillary venules bearing unusually high-walled endothelia (Gowans and Knight 1964) and named high endothelial venules (H EVs) (Stamper and Woodruff 1976). The cell surface molecules mediating the highly organ-specific binding of lymphocytes to HEVs prior to transmigration through the vessel wall have been called lymphocyte homing receptors (Gallatin etal. 1983), and their comlementary ligands on the endothelial surface addressins (Streeter etal. 1988). Thus far, several functionally and antigenically distinct lymphocyte-HEV recognition systems governing the homing of lymphocytes to peripheral lymph nodes, mucosal lymphoid organs (Peyer’s patches and appendix), and inflamed synovium have been identified (reviewed in Jalkanen et al. 1986a; Gallatin et al. 1986; Yednock and Rosen 1989).

Directed migration of lymphocytes from blood into lymph nodes and gut‐associated lymphatic tissue, also referred to as homing, is subject to change following activation. Lymphocyte migration into lymphoid organs in vivo and binding to high endothelial venules in vitro is largely suppressed after short‐term stimulation with phorbol esters. The observed functional alterations were correlated with changes in the expression of three putative homing receptors. LECAM‐1 (MEL‐14 antigen), LPAM‐1/2 (α4‐integrin) and the murine CD44 (Pgp‐1, H‐CAM, Hermes‐antigen equivalent) upon different modes of cellular activation. Expression of LECAM‐1 (gp90 MEL‐14), a lymphocyte adhesion molecule implicated in targeting extravasation into lymph nodes, was found to be lost almost completely within minutes after protein kinase C activation LECAM‐1 re‐expression occurred within less than 24 h. Rapid loss of LECAM‐1 was also observed after calcium ionophores whereas anti‐CD3 or concanavalin A elicited a gradual and heterogeneous loss of LECAM‐1 becoming delectable after several hours only. A number of cytokines tested were not able to induce alterations in LECAM‐1 expression. In contrast, expression of LPAM‐1/2 (α4‐integrin) and CD44 (Pgp‐1, H‐CAM), two adhesion molecules supposed to direct extravasation into Peyer's patches, remained stable for hours after every stimulus tested; CD44 expression gradually increased 24 h after mitogenic activation, whereas a small reduction only was observed for the expression of the α4‐chain under certain conditions. Thus, reduced extravasation of lymphocytes into Peyer's patches after activation is not due to a decline in the surface density of LPAM‐1/2 α‐chain or CD44 whereas alterations in migration into lymph nodes parallel the expression of LECAM‐1.

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.

Selection of monoclonal antibodies for differential reactivity with benign and malignant melanocytic lesions has led to the identification of molecules which may be involved in the development of metastases. Based on the observed alterations in the antigenic profile we propose a scheme representing the tumor progression of melanocytes to metastatic melanoma.

The 89-kDa cell surface glycoprotein, P3.58, is detectable on advanced human melanomas in situ but not on benign melanocytes or early melanomas. cDNA cloning of P3.58 from melanoma cells was accomplished by screening a lambda zap expression vector library with monoclonal antibodies produced against the denatured antigen. Nucleotide sequencing of the clones revealed that P3.58 is identical to the intercellular-adhesion molecule 1. No qualitative differences in P3.58 mRNA species could be seen between melanoma cells and hematopoietic cells and no differences in gene organization were observed between peripheral blood leukocytes and melanoma cells. Inspection of the deduced amino acid sequence of P3.58 indicated the presence of the consensus sequence characteristic for complement-binding proteins. The acquisition of this cell-adhesion molecule during the process of tumor progression is speculated to contribute to the development of metastasis in melanoma.

Lymphocytes home to various lymphoid organs by adhering to and migrating through specialized high endothelial venules (HEV). The murine cell surface heterodimer LPAM‐1 is involved in the homing of lymphocytes to mucosal sites (Peyer's patches). LPAM‐1 has an alpha subunit (alpha 4m) analogous to the alpha chain of the human integrin molecule VLA‐4. Here we show that the LPAM‐1 beta subunit (beta p) is immunochemically and biochemically distinct from previously defined integrin beta subunits, suggesting that beta p represents a novel integrin beta subunit. Depending on the cellular source two alternative beta subunits, beta p and integrin beta 1, can be isolated in association with alpha 4m. Therefore, alpha 4m is the common subunit of the unique integrin LPAM‐1 (alpha 4m beta p) and of the heterodimer LPAM‐2 (alpha 4m beta 1), which is analogous to VLA‐4. Antibody‐blocking experiments suggest that, in addition to LPAM‐1, LPAM‐2 is also involved in the organ‐specific adhesion of lymphocytes to Peyer's patch HEV.

Selection of monoclonal antibodies for differential reactivity with benign and malignant melanocytic lesions has led to the identification of molecules which may be involved in the development of metastases. Based on the observed alterations in the antigenic profile we propose a scheme representing the tumor progression of melanocytes to metastatic melanoma.

The development of lymphocytes and blood cells from hematopoietic precursors is one of the most remarkable and mysterious programs in developmental biology. The prime characteristics of this complex vertebrate system that make them accessible for experimental study are the known functional properties of the mature cell types, the identification of phenotypic markers expressed by these cells during several stages of their development, and most importantly, the ability to obtain these cells in suspension, mark them and separate out specific subsets, and place these fractions back in vivo to study their unique developmental and functional capacities. Here, we present studies on four aspects of mouse hematolymphoid development. The first aspect is the purification of mouse hematopoietic stem cells and the identification of a multipotent (nonstem cell) precursor population and several classes of lineage-restricted progenitors. The second is the differentiation sequence of thymic progenitors and the demonstration that loss of mature anti-self...