Plunkett, M.L. & Springer, T.A. Purification and characterization of the lymphocyte function-associated-2 (LFA-2) molecule.
J. Immunol. 136, 11, 4181-4187 (1986).
AbstractThe lymphocyte function-associated-2 (LFA-2) molecule, equivalent to CD2 and the E rosette receptor, was purified by MAb affinity chromatography from the Jurkat T lymphoma cell line. Jurkat was selected for its high level of expression of 1.0 X 10(5) sites/cell. A two-site radioimmunometric assay was developed to monitor purification. From 50 g of packed cells, 230 micrograms of LFA-2 was obtained with 65% yield of antigenic activity with a purification factor of 13,000. A major component of 58,000 and 54,000 was obtained that corresponded to LFA-2 antigenic activity as shown by immunoblotting and immunoprecipitation. The doublet was resolved by 2D IEF-SDS-PAGE into components of pI = 5.5 and 5.6. Smaller amounts of lower Mr components were also seen. All these components appeared related by processing or proteolytic breakdown, as shown by Cleveland peptide mapping. The LFA-2 deoxycholate complex had an apparent Mr of 68,000 by gel filtration, suggesting it was monomeric. Purified LFA-2 inhibited rosetting of T lymphocytes with sheep E, and addition to preformed rosettes caused their disruption. Inhibitory activity was absorbed by sheep E. This is the first evidence that the CD2/LFA-2 molecule can directly bind to sheep E. Purified LFA-2 should be useful for the further biochemical and functional characterization of this molecule.
Plunkett_1986_4750.pdf Miller, L.J., Schwarting, R. & Springer, T.A. Regulated expression of the Mac-1, LFA-1, p150,95 glycoprotein family during leukocyte differentiation.
J. Immunol. 137, 9, 2891-2900 (1986).
AbstractThe regulation of Mac-1, LFA-1, and p150,95 expression during leukocyte differentiation was examined. LFA-1 was present on almost all cell types studied. Both Mac-1 and p150,95 were present on the more mature cells of the myelomonocytic series, but only p150,95 was detected on some B cell lines and cloned cytotoxic T lymphocytes. Phorbol myristate acetate (PMA) stimulation of B chronic lymphocytic leukemia cells dramatically increased p150,95 expression. The resultant Mac-1, LFA-1, p150,95 phenotype resembled hairy cell leukemia, a B cell plasmacytoid leukemia. The promonocytic cell line U937 and the promyeloblastic cell line HL-60 expressed only LFA-1. Monocytic differentiation of U937 cells was stimulated by PMA, and induced the concomitant expression of Mac-1 and p150,95, with more p150,95 induced than Mac-1. Granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulation of U937 cells gave similar results. PMA-stimulated monocytic differentiation of the HL-60 cell line also induced expression of both Mac-1 and p150,95. The number of p150,95 molecules on PMA-stimulated U937 and HL-60 cells were 5 X 10(5) and 3 X 10(5), respectively. Retinoic acid stimulated myeloid differentiation of HL-60 cells and induced expression of both Mac-1 and p150,95. These cells acquired a Mac-1, LFA-1, p150,95 profile that resembled that of granulocytes, with more Mac-1 than p150,95 induced. GM-CSF stimulation of HL-60 cells induced a similar Mac-1 and p150,95 phenotype. The contributions of Mac-1, LFA-1, and p150,95 to aggregation of PMA-differentiated U937 cells were assessed. Monoclonal antibodies to the beta subunit and the LFA-1 alpha subunit, but not those to p150,95 or Mac-1 alpha subunit, inhibited this homotypic adherence.
Miller_1986_4772.pdf Rothlein, R. & Springer, T.A. The requirement for lymphocyte function-associated antigen 1 in homotypic leukocyte adhesion stimulated by phorbol ester.
J. Exp. Med. 163, 5, 1132-1149 (1986).
AbstractLymphocytes become adherent and aggregate after stimulation with phorbol esters such as PMA. Time-lapse video showed that aggregating cells were motile and exhibited vigorous pseudopodial movements. Adhesion sites were initiated between pseudopodia of neighboring cells, and then moved to the uropod. PMA-stimulated aggregation by EBV-transformed B cell lines, SKW-3 (a T cell line), differentiated U937 (a monocytic line), and blood lymphocytes was inhibited by mAbs to LFA-1. A number of different mAb to the LFA-1 alpha and beta subunits and F(ab')2 and Fab' fragments inhibited aggregation. Furthermore, lymphoblasts from normal individuals, but not from LFA-1-deficient patients, aggregated in response to PMA. These findings suggest LFA-1 is critically involved in stimulated lymphocyte adhesion. LFA-1 expression was not increased by PMA stimulation, showing that other mechanisms regulate LFA-1-dependent adherence. LFA-1-deficient patient cells were able to coaggregate with LFA-1+ cells, showing that aggregation is not mediated by like-like interactions between LFA-1 molecules on opposite cells. Aggregation was Mg+2-dependent, inhibited by cytochalasin B, and was reversed when LFA-1 mAb was added to preformed aggregates. Previous findings suggesting that LFA-1 is important in a wide variety of leukocyte functions are elucidated by this work, which shows that LFA-1 is a general leukocyte cell adhesion molecule, the activity of which is regulated by cell activation.
Rothlein_1986_4513.pdf Haskard, D., Cavender, D., Beatty, P., Springer, T.A. & Ziff, M. T lymphocyte adhesion to endothelial cells: Mechanisms demonstrated by anti-LFA-1 monoclonal antibodies.
J. Immunol. 137, 9, 2901-2906 (1986).
AbstractAdhesion of lymphocytes to vascular endothelium is the first event in the passage of lymphocytes into a chronic inflammatory reaction. To investigate molecular mechanisms of T-EC adhesion, monoclonal antibodies (Mab) against T cell surface antigens have been tested for inhibition of binding. Baseline and phorbol ester-stimulated adhesion were strongly inhibited by either Mab 60.3 (reactive with the beta-chain of the LFA-1, OKM1, and p150,95 molecules) or by Mab TS 1/22 (specific for the alpha-chain of LFA-1). Although the increased binding of phorbol ester-stimulated lymphocytes was inhibited by anti-LFA-1 antibody, there was no increased expression of LFA-1 on phorbol ester-stimulated T cells, as determined by FACS analysis. Maximal inhibition of unstimulated and phorbol ester-stimulated T-EC adhesion was seen at Mab concentrations of 1 microgram/ml. In contrast, LPS- and IL 1-enhanced T-EC adhesion were only weakly inhibited by these antibodies. Mab 60.3 and TS 1/22 did not stain either unstimulated EC or LPS- or IL 1-stimulated EC, as measured by FACS analysis; moreover, preincubation of EC alone with these antibodies did not lead to inhibition of T-EC binding. Adhesion was not affected by Mab against the sheep erythrocyte receptor (LFA-2), a nonpolymorphic HLA class 1 framework antigen, or against LFA-3, the alpha-chain of OKM1, or the alpha-chain of p150,95. These results suggest that the mechanism of binding of lymphocytes to unstimulated endothelium differs from that to stimulated endothelium. LFA-1 appears to be an important adhesion-related molecule for binding to unstimulated endothelium. However, the increased lymphocyte adhesion to IL 1- or LPS-stimulated EC observed in these experiments appears to be relatively independent of LFA-1. The increased adhesion to stimulated EC could be due either to an increase in the avidity or the density of the EC receptor molecules ordinarily involved in unstimulated T-EC binding or to the formation of alternative receptors on the stimulated EC that are not present on unstimulated cells.
Haskard_1986_5385.pdf Shaw, S., et al. Two antigen-independent adhesion pathways used by human cytotoxic T cell clones.
Nature 323, 6085, 262-264 (1986).
AbstractCell-cell adhesion is essential for many immunological functions, including interaction of cytotoxic T lymphocytes (CTLs) with their targets. We have explored CTL-target interactions using well-characterized cloned human CTLs. Conjugate formation between these CTLs and many antigen-negative targets is almost as efficient as with specific target cells, but does not lead to target-cell lysis. Thus, on specific target cells, adhesion by antigen-independent pathways may occur concurrently with or precede antigen recognition. The molecules LFA-1, CD2 (T11, LFA-2) and LFA-3 have been shown to be involved in human CTL conjugation with and lysis of specific target cells. Here we describe monoclonal antibody inhibition studies using individual monoclonal antibodies and mixes which demonstrate (1) that LFA-1, CD2 and LFA-3 are involved in antigen-independent conjugate formation; and (2) suggest that CD2 and LFA-3 are involved in one pathway and LFA-1 in another. We confirmed the existence of distinct pathways by the demonstration that LFA-1-dependent adhesion requires divalent cations and is temperature-sensitive whereas CD2- and LFA-3-dependent adhesion does not require divalent cations and is temperature-insensitive. Together with previous data, our studies suggest that CD2 on the effector interacts with LFA-3 as its ligand on targets.
Shaw_1986_4774.pdf