Tumor-specific responses in lymph nodes draining murine sarcomas are concentrated in cells expressing P-selectin binding sites.

Tumor-draining lymph node (TDLN) cells develop substantial antitumor activity after activation on immobilized alphaCD3 and culture in low-dose IL-2. This study found that the minor subset of TDLN T cells expressing binding sites for the adhesion receptor P-selectin (Plig(high) T cells) produced T lymphoblasts with the most tumor-specific IFN-gamma synthesis in vitro and antitumor activity following adoptive transfer in vivo. The Plig(high) T cells constituted <25% of the cells with the phenotype of recently activated cells including high levels of CD69, CD44, or CD25, and low levels of CD62L. The cultured Plig(high) TDLN were 10- to 20-fold more active against established pulmonary micrometastases than cultured unfractionated TDLN, and >30-fold more active than cultured TDLN cells depleted of the Plig(high) fraction before expansion (Plig(low) cells). Tumor-specific IFN-gamma synthesis in vitro paralleled the antitumor activities of the cultured fractions in vivo, implying that increased Tc1 and Th1 effector functions contributed to the tumor suppression. Neither nonspecific interaction with the P-selectin chimera used for sorting nor endogenous costimulatory activity in the Plig(high) fraction accounted for the marked increase in antitumor activities after culture. The cultured Plig(high) fraction contained a variety of potential effector cells; however, the CD8 and CD4 subsets of alphabeta T cells accounted for 95-97% of its antitumor activity. The authors propose that P-selectin sorting increased antitumor activities by concentrating Tc1 and Th1 pre-effector/effector cells before culture.

Adhesion molecule expression in CD5-negative/CD10-negative chronic B-cell leukemias: comparison with non-Hodgkin's lymphomas and CD5-positive B-cell chronic lymphocytic leukemia.

The classification of CD5-negative/CD10-negative chronic B-cell leukemias (CD5-/CD10- CBL) can be problematic. Most of these cases may represent leukemic non-Hodgkin's lymphoma (NHL) other than B-cell chronic lymphocytic leukemia (BCLL); nonetheless, some investigators still advocate the term "CD5-negative BCLL." Because adhesion molecule (AdMol) expression patterns reflect the biology of lymphoid neoplasms, we studied a series of 106 B-cell lymphoproliferative disorders, including CD5+ BCLL (n = 56), NHL other than BCLL (n = 35), and CD5-/CD10- CBL (excluding hairy cell leukemia and prolymphocytic leukemia) with no prior history of NHL (n = 15) for expression of components of the very late antigen-4 complex (alpha4/beta1 integrin (CD49d/CD29)), components of the mucosal addressin-cell adhesion molecule receptor (alpha4(CD49d)/beta7 integrin), and L-selectin (CD62L). CD62L expression was significantly greater in CD5+ BCLL than in NHL (P < .001). Conversely, CD29, CD49d, and beta7-integrin expression were significantly greater in NHL than in CD5+ BCLL (P < .001 for each marker). These differences persisted when only blood and bone marrow samples were analyzed, with the exception of differences in CD62L expression, which approached, but did not reach, statistical significance (P = .08). The group of CD5-/CD10- CBL displayed an AdMol profile similar to NHL and was significantly different than CD5+ BCLL in expression of beta7 integrin, CD29, CD49d, and CD62L (P range < .001-.011). In summary, CD5-/CD10- CBL display an AdMol profile resembling NHL and significantly different from CD5+ BCLL, supporting the growing notion that "CD5-negative BCLL" generally represents leukemic NHL rather than a variant of true CD5+ BCLL.

Effects of tumor necrosis factor-alpha on the in vitro maturation of tumor-reactive effector T cells.

Culture methods that enhance the anti-tumor reactivity of primed T cells would be important in adoptive immunotherapy of cancer. Using several different syngeneic murine tumor models, the authors evaluated the effects of tumor necrosis factor-alpha (TNF-alpha) exposure on tumor-draining lymph node (TDLN) cells during in vitro activation. Mice were inoculated with weakly immunogenic (i.e., MCA 205, MCA 207 sarcoma) or the poorly immunogenic (i.e., D5 melanoma) tumor cells, and TDLN cells were harvested 9 or 10 days later for activation by an anti-CD3/interleukin-2 culture procedure. Human recombinant TNF-alpha (25 ng/mL) added during the activation culture resulted in a two-fold increase in interferon-gamma release (type 1 response) and a significant reduction of interleukin-10 (type 2 response) after tumor antigen stimulation. In an adoptive transfer model, TNF-alpha-cultured TDLN cells mediated significantly greater regression of established tumor than did TDLN cells cultured in the absence of TNF-alpha in five of five experiments. Neutralization of interleukin-10 monoclonal antibody further augmented the therapeutic efficacy of TNF-alpha-cultured TDLN cells. These studies document the ability of TNF-alpha to selectively promote a type 1 over a type 2 response in a bulk population of tumor-primed T cells during in vitro activation.

Lymphocyte-endothelial cell adhesive interactions in lung immunity: lessons from the murine response to particulate antigen.

The adhesive interaction between lymphocytes and lung endothelial cells presents an attractive arena for the development of novel therapeutic agents to modify pathologic pulmonary immune responses. The conceptual basis for choosing molecular targets to modulate this adhesive interaction derives, in large part, from results of murine experimental model systems of the pulmonary immune response. This article reviews one such model, the response of primed C57BL/6 mice to the particulate antigen sheep erythrocytes. Novel data are presented on the effect of a blocking anti-alpha(4) integrin monoclonal antibody on lung leukocyte and lymphocyte subset accumulation after intratracheal (IT) antigen challenge. Results from this model system have indicated that lymphocytes may use either the endothelial selectins or alpha(4) integrin as independent pathways to initiate recruitment into the lungs.

Alpha(1,3)-fucosyltransferase VII-dependent synthesis of P- and E-selectin ligands on cultured T lymphoblasts.

T lymphocytes up-regulate the synthesis of ligands for E- and P-selectin during proliferative responses in vivo and in vitro. Previous studies from our laboratories indicated that the alpha(1,3)-fucosyltransferase FucT-VII regulates the synthesis of E-selectin ligands and sialylated Lewis(x)-related epitopes (sLe(x)-related epitopes) in human T lymphoblasts. The current report shows that production of both P- and E-selectin ligands is FucT-VII dependent, but peak synthesis of each occurs at different levels of fucosyltransferase activity in intact cells. In brief, FucT-VII mRNA levels were higher in cultured T lymphoblasts expressing sLe(x)-related epitopes and both selectin ligands than in cells expressing P-selectin ligands alone. However, synthesis of the epitopes and both selectin ligands required the FucT-VII enzyme in transfected Molt-4 cells. In contrast, neither constitutive nor transfection-enhanced levels of the FucT-IV enzyme generated active P-selectin ligands in these lines. In addition, targeted deletion of the FucT-VII gene in mice markedly inhibited the synthesis of both P- and E-selectin ligands during blast transformation in vitro. Finally, the optimal synthesis of active P-selectin ligands occurred at lower level of FucT-VII activity than required for synthesis of equally active E-selectin ligands in both cultured T lymphoblasts and FucT-VII transfectants. Consequently, the FucT-VII enzyme is essential for the synthesis of both P- and E-selectin ligands by T lymphoblasts, and its activity determines whether P-selectin ligands are expressed alone or in conjunction with E-selectin ligands and sLe(x)-related epitopes on human T cells.

Interleukin 12 and interleukin 4 control T cell adhesion to endothelial selectins through opposite effects on alpha1, 3-fucosyltransferase VII gene expression.

The alpha1,3-fucosyltransferase, FucT-VII, is crucial for the formation of ligands for all three selectins, and its expression regulates the synthesis of these ligands. Short-term polarized T helper (Th)1, but not Th2 or naive CD4(+) T cells, can home to sites of inflammation, but the molecular basis for this difference has remained unclear. Here we show that naive CD4(+) T cells do not express FucT-VII and fail to bind vascular selectins. We also show that when CD4(+) T cells are activated in the presence of the Th1 polarizing cytokine interleukin (IL)-12, levels of FucT-VII mRNA and binding to E- and P-selectin are significantly augmented. In contrast, activation of CD4(+) T cells in the presence of IL-4, a Th2 polarizing cytokine, inhibited FucT-VII expression and binding to vascular selectins. T cell activation upregulated expression of the Core2 transferase, C2GnT, equivalently regardless of the presence or absence of polarizing cytokines. These data indicate that the selective ability of Th1 cells, as opposed to Th2 cells or naive CD4(+) T cells, to recognize vascular selectins and home to sites of inflammation is controlled principally by the expression of a single gene, FucT-VII.

Endothelial selectins and alpha4 integrins regulate independent pathways of T lymphocyte recruitment in the pulmonary immune response.

The cell adhesion molecules (CAMs) required for T lymphocyte recruitment during pulmonary immune responses have not been defined. Our laboratories recently reported that intratracheal (IT) challenge of sensitized mice with SRBC induced prolonged expression of vascular P-selectin, E-selectin, and VCAM-1, particularly in areas of mononuclear leukocyte infiltration. A surge in the number of circulating T lymphocytes expressing selectin ligands preceded the peak accumulation of T cells in the lung. In addition, a significant percentage of the T cells recovered from the lung expressed selectin ligands as well. The current study demonstrates that cultured T lymphoblasts use both selectin ligands and alpha4 integrins to enter the airspace and interstitium during the response to SRBC. Fluorescently labeled T lymphoblasts, derived via activation on CD3 and growth in low dose IL-2, showed inflammation-specific recruitment into lungs harvested 24 h after cell infusion. Their flux paralleled the accumulation of host lymphocytes in the lung, with both peaking 2 to 4 days after SRBC challenge. Trafficking studies conducted over a 24-h period during peak lymphocyte accumulation in the lungs revealed preferential recruitment of labeled T lymphoblasts expressing P- and E-selectin ligands. In addition, mAb blockade of the alpha4 integrins and targeted deletion of an alpha(1,3)fucosyltransferase essential for selectin ligand synthesis each reduced labeled T lymphoblast trafficking to a significant degree. Furthermore, alpha4 integrin blockade reduced the trafficking of the selectin ligand-deficient cells into the airspace, confirming that its contribution is in part independent from the vascular selectins. These findings imply that both selectin ligands and alpha4 integrins participate in T lymphoblast recruitment during the pulmonary immune response to IT SRBC.

Dimerization of P-selectin glycoprotein ligand-1 (PSGL-1) required for optimal recognition of P-selectin.

Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with alpha1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin.

An sLex-deficient variant of HL60 cells exhibits high levels of adhesion to vascular selectins: further evidence that HECA-452 and CSLEX1 monoclonal antibody epitopes are not essential for high avidity binding to vascular selectins.

Selectins are carbohydrate-binding cell adhesion molecules that play a key role in the initiation of inflammatory responses. Several studies have suggested that the sialylated, fucosylated tetrasaccharide sialyl Lewis X (sLex) is an important component of leukocyte ligands for E- and P-selectin. We have identified a stable variant of the HL60 cell line, HL60var, which displays a nearly complete absence of staining with several mAb directed against sLex and/or sLex-related structures. HL60var also exhibits a concomitant increase in reactivity with mAb directed against the unsialylated Lewis X (Lex/CD15) structure. Despite this sLex deficiency, HL60var binds well to both E- and P-selectin. No significant differences in expression of alpha1,3-fucosyltransferases, C2GnT (Core2 transferase), or P-selectin glycoprotein ligand-1 between HL60var and typical sLex(high) HL60 cells were detected. Although the precise molecular basis for the sLex(-/low) phenotype of HL60var remains uncertain, flow cytometric analysis with the sialic acid-specific Limax flavus lectin revealed a sharp reduction in HL60var surface sialylation. Thus, the loss in mAb reactivity may result from a loss of sialic acid residues from the mAb carbohydrate epitope. However, binding of HL60var to E- and P-selectin remains sensitive to neuraminidase treatment. Taken together, these data indicate that high levels of surface sLex and/or related epitopes are not essential for interactions with vascular selectins, implying that as yet unidentified sialylated, fucosylated structures serve as physiologically relevant ligands for E- and P-selectin.

Lymphocyte recruitment and the kinetics of adhesion receptor expression during the pulmonary immune response to particulate antigen.

The selectins and beta2 integrins participate in the recruitment of neutrophils in acute pulmonary inflammation. However, the cell adhesion receptors that mediate lymphocyte trafficking into the lung have not been defined. This study examined the relationship between cell adhesion molecules on the pulmonary vasculature and on lymphocytes recovered from the lung during a pulmonary immune response to intratracheal (I.T.) sheep red blood cells (SRBCs) in sensitized C57BL/6J mice. Silver-enhanced immunogold staining and reverse transcriptase polymerase chain reaction of lung tissues revealed sustained induction of VCAM-1, E-selectin, and P-selectin on the pulmonary vasculature for up to 7 days after I.T.-SRBC challenge. Neither the MECA 79 nor MECA 367 antigens were induced on the pulmonary vasculature during this period. In the peripheral blood, both CD4 and CD8 T-cell subsets showed an initial increase in P-selectin ligand expression after I.T.-SRBC challenge. The number of P-selectin ligand-positive T cells in the peripheral blood fell as T cells with both P-selectin and, to a lesser extent, E-selectin ligands accumulated in the bronchoalveolar lavage fluid. We conclude that I.T.-SRBC challenge in sensitized mice elicits prolonged synthesis of P-selectin, E-selectin, and VCAM-1 by the lung vasculature as well as selectin ligand synthesis by responding T cells. Furthermore, the entry of selectin-ligand-positive T cells into the circulation and their accumulation in the bronchoalveolar lavage fluid indicates that these receptors may contribute to T cell recruitment. Finally, VCAM-1 on the vasculature may also participate; however, the vascular addressins, required for homing to peripheral and mucosal lymphoid organs, are not essential for T-cell entry into the lung following I.T.-SRBC challenge.

Expression of leukocyte fucosyltransferases regulates binding to E-selectin: relationship to previously implicated carbohydrate epitopes.

E-selectin is a carbohydrate-binding endothelial cell adhesion molecule that reportedly interacts with several related sialylated and fucosylated carbohydrates. The activity of leukocyte alpha1,3-fucosyltransferases (FucT-IV or FucT-VII) is an essential step in the synthesis of E-selectin ligands. Using a panel of stably transfected hemopoietic cell lines, we have investigated the role of alpha1,3-fucosyltransferases in generating E-selectin ligands, and the relationship between adhesion to E-selectin and expression of mAb-defined carbohydrates. Expression of FucT-VII was always sufficient for binding to E- and P-selectin, while the ability of FucT-IV to construct E-selectin ligands varied among different cell types. Furthermore, FucT-IV was unable to support any binding to P-selectin in a lymphoid cell line, even when expressed at levels equivalent to those in myeloid cells. FucT-IV expression generated high levels of surface Le(x)/CD15 and CDw65, whereas expression of FucT-VII correlated with a subset of mAb-defined sialyl Lewis X (sLex)-like structures. FucT-IV-associated epitopes were present on both binding and nonbinding cells, whereas all cells that expressed FucT-VII-associated epitopes bound E-selectin. However, treatment of HL60 cells with neuraminidase destroyed FucT-VII-associated epitopes at a faster rate than E-selectin binding sites. Surface expression of a subset of mAb-defined sLex-like carbohydrates is therefore a good marker for high levels of FucT-VII activity, but these carbohydrates are not themselves required for recognition of E-selectin.

P-selectin glycoprotein ligand-1 is essential for adhesion to P-selectin but not E-selectin in stably transfected hematopoietic cell lines.

P-selectin (CD62P) is a member of the selectin family of adhesion molecules involved in the regulation of leukocyte traffic. P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like molecule that is thought to be a primary ligand for P-selectin. The interaction of P-selectin with PSGL-1 results in leukocyte rolling and recruitment of leukocytes to sites of inflammation and tissue injury. However, expression of PSGL-1 protein alone is insufficient for binding to P-selectin. Several posttranslational modifications of PSGL-1, including sialylation, sulfation, and fucosylation by alpha 1,3-fucosyltransferase(s) (FucT), are required for functional interaction with P-selectin. Recently, several groups have reported that PSGL-1 might also serve as a ligand for E-selectin. Differential posttranslational modifications of PSGL-1 may determine whether it can interact with either P- or E-selectin or both. To determine whether PSGL-1 is essential for adhesion to P- or E-selectin, we have constructed and analyzed a panel of stably transfected K562 cells. K562 cells express FucT-IV but not FucT-VII or PSGL-1, and do not bind to either E- or P-selectin. K562 cells transfected with PSGL-1 cDNA also did not bind to either P- or E-selectin. Binding to P-selectin occurred only when K562 cells were cotransfected with both FucT-VII and PSGL-1. In contrast, expression of FucT-VII alone was sufficient for E-selectin binding. These data demonstrate that expression of PSGL-1 is not required for adhesion of a stably transfected hematopoietic cell line to E-selectin, and suggest that FucT-IV alone cannot properly modify PSGL-1, expressed in transfected K562 cells, to bind P-selectin.

Adhesion of memory lymphocytes to vascular cell adhesion molecule-1-transduced human vascular endothelial cells under simulated physiological flow conditions in vitro.

The accumulation of mononuclear leukocytes is an early and persistent finding in atherosclerotic plaques. These mononuclear leukocytes are mostly monocyte-derived, but up to 20% are lymphocytes, predominantly CD4+ CD45RO+ (memory) T cells. To evaluate the potential of adenovirus vectors for studies of mononuclear leukocyte recruitment in vitro, we studied the effects of adenovirus vectors per se on human umbilical vein endothelial cells (HUVECs), a well-characterized in vitro model of vascular endothelium. A recombinant adenovirus containing the seven-domain isoform of rabbit vascular cell adhesion molecule-1 (rVCAM-1) was constructed and used to study lymphocyte adhesion under defined laminar flow conditions in transduced HUVEC monolayers. No increase in basal HUVEC surface expression of the inducible endothelial adhesion molecules and markers of activation, E-selectin and VCAM-1, was noted across a broad range of multiplicity of infection. A modest dose-dependent increase in surface intercellular adhesion molecule-1 expression was detectable by flow cytometry at an MOI of > 30 plaque-forming units per cell. Under defined laminar flow from 1.5 to 0.5 dyne/cm2, the adenovirus vector carrying rVCAM-1 mediated stable adhesion of both a Jurkat T-cell line and primary human CD4+ CD45RO+ (memory) T cells. Monoclonal antibodies to alpha 4-integrin or rVCAM-1 abolished adhesion, whereas monoclonal antibodies to CD18 or P-selectin had no effect. We conclude that adenoviral gene transfer in useful for studies of VCAM-1-dependent leukocyte adhesion in vitro and that endothelial expression of VCAM-1 alone, in the absence of over endothelial cell activation, is sufficient under simulated physiological flow conditions to support adhesion of memory T cells, the predominant lymphocyte subset in atherosclerotic plaque.

The fucosyltransferase FucT-VII regulates E-selectin ligand synthesis in human T cells.

Selectin-ligands on T cells contribute to the recruitment of circulating cells into chronic inflammatory lesions in the skin and elsewhere. This report provides the first evidence that a single fucosyltransferase, termed FucT-VII, controls the synthesis of E-selectin ligands in human T-lymphoblasts. The FucT-IV transferase (the ELFT enzyme), in contrast constructs lower avidity E-selectin ligands and requires enzyme levels found only in myeloid cells. Treatment of Jurkat cells with phorbol myristate acetate increased the expression of sialylated Lewis(x)-related sLe(x)related epitopes and induced the synthesis of E-selectin ligands functional at physiologic levels of linear shear-stress. Northern analysis revealed a parallel increase in the steady-state levels FucT-VII mRNA, but there were no increases in the two other leukocyte-associated fucosyltransferases (FucT-IV and VI). The stable transfection of the FucT-VII gene into Jurkat cells induced high levels of the sLe(x)-related epitopes and the synthesis of E-selectin ligands which equal or exceeded the avidity of those on circulating lymphocytes. The growth of T-lymphoblasts under conditions which induced expression of the sLe(x,a) epitopes increased the level of FucT-VII mRNA, the synthesis of sialylated-Lewis(x) structures by cell-free extracts and the synthesis of E-selectin ligands equal in avidity to those on FucT-VII transfectants. In contrast, neither the mRNA levels nor activities of the FucT-IV and VI enzymes increased in association with E-selectin ligand synthesis in T-lymphoblasts. Myeloid cell lines, unlike lymphoblasts, expressed high levels of both the FucT-VII and IV enzymes in conjunction with E-selectin ligands raising the possibility that both enzymes contributed to ligand synthesis. FucT-IV transfected Jurkat cells synthesized low avidity ligands for E-selectin but only in association with CDw65 (VIM-2) carbohydrate epitope. Only blood neutrophils and myeloid cell lines expressed this epitope at the levels associated with E-ligand synthesis in the transfectants. In contrast, native Jurkat cells, blood monocytes, blood lymphocytes, and cultured T-lymphoblasts expressed low levels or none. We conclude that FucT-VII is a principal regulator of E-selectin ligand synthesis in human T-lymphoblasts while both FucT-VII and FucT-IV may direct ligand synthesis in some myeloid cells.

Sialylated O-glycans and L-selectin sequentially mediate myeloid cell rolling in vivo.

Leukocyte rolling precedes firm adhesion and emigration in inflammatory cell recruitment. Both P-selectin, an endothelial lectin that binds to sialylated O-glycans containing sialyl-Lewisx (sLex) on the granulocyte surface, and leukocyte L-selectin have been shown to mediate leukocyte rolling in vivo. Here, we investigate rolling of isolated human neutrophils (PMN), HL-60 promyelocytes, and an L-selectin-transfected cell line (300.19-L) during trauma-induced inflammation in rat mesenteric venules. HL-60 cells, which express no L-selectin but abundant sLex, rolled effectively immediately after abdominal surgery. HL-60 cell rolling was almost completely abolished by pretreatment with sialidase or monoclonal antibody (MoAb) AM-3 recognizing sLex, and was reduced by about 80% by O-sialoglycoprotein-endopeptidase (OSGP). By contrast, 300.19-L cells rolled poorly immediately after surgery but rolled well between 40 and 120 minutes after surgery. Their rolling was completely inhibited by the blocking L-selectin MoAb LAM1-3, but not by a binding control MoAb. PMN express both L-selectin and clustered, sialylated glycoproteins including P-selectin glycoprotein ligand-1 (PSGL-1). PMN showed effective rolling at all times, which was abolished by sialidase or MoAb AM-3 pretreatment during the first 30 minutes after surgery, but not later, when PMN rolling was largely L-selectin-dependent. We conclude that in trauma-induced inflammation, a two-step mechanism accounts for most of myeloid cell rolling, which initially requires O-glycans and subsequently depends on L-selectin function.

Regulation of L-selectin mRNA in Jurkat cells. Opposing influences of calcium- and protein kinase C-dependent signaling pathways.

L-Selectin initiates leukocyte attachment to venular endothelium during lymphocyte recirculation through lymph nodes, leukocyte recruitment into sites of inflammation, and the hematogenous spread of lymphoid malignancies. The density of L-selectin at the cell surface is a major determinant of binding activity and entry into tissues. Post-transcriptional shedding is one control mechanism; however, the extent and physiologic relevance of pre-translational regulation has not been defined. The current study shows that mitogen-/IL-2-driven proliferation of human T cells first increased then markedly decreased the expression of L-selectin on the blast population. The prevalence of specific mRNA showed parallel changes, implying that receptor density is controlled, in part, at the pretranslational level. We used the IL-2-independent Jurkat cell line to determine whether signaling through C-type protein kinases and intracellular calcium regulated L-selectin mRNA directly. Selective pharmacologic activation of these pathways with phorbol esters and calcium ionophore, respectively, resulted in opposite effects on both L-selectin density and mRNA levels. Phorbol esters induced receptor shedding followed by progressive increases in L-selectin density and steady state levels of mRNA. Addition of a calcium ionophore with the phorbol ester blocked both the reexpression of surface receptor and the increase in mRNA. Treatment with ionophore alone resulted in a steady decline in L-selectin expression and mRNA levels. Cyclosporin A, a specific inhibitor of calcineurin, blocked the impact of ionophore on both basal and phorbol-induced levels of L-selectin mRNA. Ionophore alone did not induce apoptosis, significantly alter cell cycle kinetics, or increase transcription of the IL-2 gene under conditions that suppressed L-selectin. Thus, calcineurin seems to be a proximal enzyme in a novel regulatory cascade that suppresses L-selectin expression independent of its known effects on proliferating T cells. In light of the findings in Jurkat, we propose that the protein kinase pathway up-regulates L-selectin mRNA and surface expression early in mitogen-driven T cell proliferation. Chronic elevation of intracellular calcium in repeatedly stimulated T cells then down-regulates expression at the pretranslational level through prolonged activation of calcineurin.

Monocyte-endothelial adhesion in chronic rheumatoid arthritis. In situ detection of selectin and integrin-dependent interactions.

Blood monocytes are the principal reservoir for tissue macrophages in rheumatoid synovitis. Receptor-mediated adhesive interactions between circulating cells and the synovial venules initiate recruitment. These interactions have been studied primarily in cultured endothelial cells. Thus the functional activities of specific adhesion receptors, such as the endothelial selectins and the leukocytic integrins, have not been evaluated directly in diseased tissues. We therefore examined monocyte-microvascular interactions in rheumatoid synovitis by modifying the Stamper-Woodruff frozen section binding assay initially developed to study lymphocyte homing. Specific binding of monocytes to venules lined by low or high endothelium occurred at concentrations as low as 5 x 10(5) cells/ml. mAbs specific for P-selectin (CD62, GMP-140/PADGEM) blocked adhesion by > 90% in all synovitis specimens examined. In contrast, P-selectin-mediated adhesion to the microvasculature was either lower or absent in frozen sections of normal foreskin and placenta. mAbs specific for E-selectin (ELAM-1) blocked 20-50% of monocyte attachment in several RA synovial specimens but had no effect in others. mAbs specific for LFA-1, Mo1/Mac 1, the integrin beta 2-chain, and L-selectin individually inhibited 30-40% of adhesion. An mAb specific for the integrin beta 1-chain inhibited the attachment of elutriated monocytes up to 20%. We conclude that P-selectin associated with the synovial microvasculature initiates shear-resistant adhesion of monocytes in the Stamper-Woodruff assay and stabilizes bonds formed by other selectins and the integrins. Thus the frozen section binding assay permits direct evaluation of leukocyte-microvascular adhesive interactions in inflamed tissues and suggests a prominent role for P-selectin in monocyte recruitment in vivo.

Acute biphenotypic leukaemia: immunophenotypic and cytogenetic analysis.

The incidence of acute biphenotypic leukaemia has ranged from less than 1% to almost 50% in various reports in the literature. This wide variability may be attributed to a number of reasons including lack of consistent diagnostic criteria, use of various panels of antibodies, and the failure to recognize the lack of lineage specificity of some of the antibodies used. The morphology, cytochemistry, immunophenotype and cytogenetics of acute biphenotypic leukaemias from our institution were studied. The diagnostic criteria took into consideration the morphology of the analysed cells, light scatter characteristics, and evaluation of antibody fluorescence histograms in determining whether the aberrant marker expression was arising from leukaemic blasts or differentiated bone marrow elements. Fifty-two of 746 cases (7%) fulfilled our criteria for acute biphenotypic leukaemias. These included 30 cases of acute lymphoblastic leukaemia (ALL) expressing myeloid antigens, 21 cases of acute myelogenous leukaemia (AML) expressing lymphoid markers, and one case of ALL expressing both B- and T-cell associated antigens. The acute biphenotypic leukaemia cases consisted of four major immunophenotypic subgroups: CD2+ AML (11), CD19+ AML (8), CD13 and/or CD33+ ALL (24), CD11b+ ALL (5) and others (4). Chromosomal analysis was carried out in 42/52 of the acute biphenotypic leukaemia cases; a clonal abnormality was found in 31 of these 42 cases. This study highlights the problems encountered in the diagnosis of acute biphenotypic leukaemia, some of which may be responsible for the wide variation in the reported incidence of this leukaemia. We suggest that the use of strict, uniform diagnostic criteria may help in establishing a more consistent approach towards diagnosis of this leukaemic entity. We also suggest that biphenotypic leukaemia is comprised of biologically different groups of leukaemia based on immunophenotypic and cytogenetic findings.

Regulation of fibronectin and laminin binding activity in cultured human lymphoblastic cell lines.

The current study shows that a clonal derivative of the Jurkat cell line up-regulates both the avidity and density of the alpha 6/beta 1 receptor in response to phorbol 12-myristate 13-acetate (PMA). This derivative attaches to fibronectin and, to a lesser degree, laminin constitutively. Adhesion and spreading are dramatically up-regulated following treatment with PMA. The response on fibronectin peaks within 4 hours, is insensitive to cyclohexamide, can be blocked by monoclonal antibodies (Mabs) to the beta 1 and alpha 5 subunits of the beta 1 family of integrins, and is not associated with increased expression of the alpha 5 or beta 1 epitopes at the cell surface. In contrast, the response on laminin is biphasic. The early phase parallels the response on fibronectin. The second phase peaks after 48-72 hours of treatment with PMA, is sensitive to cycloheximide, can be blocked by Mabs to the beta 1 and alpha 6 subunits, and is associated with increased expression of the alpha 6 epitope. Both the density independent and dependent responses to PMA in Jurkat cells are blocked by the protein kinase inhibitor staurosporine. The HSB-2, CEM, Molt-4, and HPB-ALL T-lymphoblastic cell lines also up-regulate attachment to fibronectin and laminin following treatment with PMA. All four lines constitutively attach to fibronectin and show rapid up-regulation of attachment following treatment with PMA. None of the lines attach to laminin prior to PMA treatment; however, specific adhesion developed after 4-120 hours of treatment. The most mature lines (Jurkat and HPB-ALL) up-regulated adhesion on laminin more rapidly than the less phenotypically mature lines (CEM, Molt-4, and HSB-2). In summary, clonal derivatives of the Jurkat cell line up-regulated attachment to laminin through protein kinase dependent increases in alpha 6/beta 1 receptor avidity and density. In addition, the expression of functional receptors for laminin is linked to developmental maturity in a series of T-lymphoblastic cell lines.