Abstract
Leukocytes use the alpha 6 beta 1 integrin to adhere to laminin based on mAb inhibition and affinity chromatography studies. This adhesion requires leukocyte stimulation with either PMA or specific cytokines, a process that has been termed "inside-out" integrin signaling. In the present study, the involvement of alpha 6 integrin structural variants in this regulated adhesion was examined using mouse macrophages. The two known alpha 6 structural variants, alpha 6A and alpha 6B, differ only in their cytoplasmic domain sequences. Using reverse transcriptase-polymerase chain reaction, we observed that macrophages express only the alpha 6A structural variant, in contrast to most cell types which express both alpha 6A and alpha 6B variants. The role of this integrin subunit in macrophage adhesion was assessed by cDNA transfection of P388D1 cells. We found that this mouse macrophage cell line does not adhere to laminin even in response to phorbol 12-myristate 13-acetate (PMA) stimulation, though it does adhere normally to fibronectin and tissue culture plastic. Subsequent analysis employing reverse transcriptase-polymerase chain reaction and immunoprecipitation of surface labeled cells revealed that this cell line expresses neither the alpha 6A nor alpha 6B integrin subunits. Stable transfection of either the chick or human alpha 6A cDNAs into P388D1 cells resulted in chimeric alpha 6A beta 1 surface expression. The alpha 6A transfectants exhibited inside-out integrin signaling because PMA stimulation markedly increased their ability to adhere to laminin but it did not increase alpha 6A beta 1 surface expression. Similar results were obtained after transfection of the human alpha 6B cDNA. Analysis of the human transfectants was facilitated by the generation of a monoclonal antibody, 2B7, that is specific for the human alpha 6 integrin subunit. These observations demonstrate that both alpha 6A beta 1 and alpha 6B beta 1 can be regulated by inside-out signaling pathways in macrophages, even though this cell type expresses only alpha 6A beta 1. The data presented also demonstrate clearly that the alpha 6A and alpha 6B cytoplasmic domains do not differ in their ability to be regulated by PMA.
Highlights
Leukocytes use thea681 integrin to adhere to lami- It is becoming apparent that the abilityof cells to interact nin based on mAb inhibition and affinity chromatog- with basement membrane componentsis physiologically regraphy studies
Using reverse transcriptase-polymerase chain reaction, woebserved that macrophages express only the a6A structural variant, in contrast to most cell typeswhich express both a6A and a6B variants. The role of this integrin subunit in macrophage adhesion wasassessed by cDNA transfection of P388D1 cells
A telltale finding in this work surface labeled cells revealed that this cell line ex- was the observation that the inductionof leukocyte adhesion presses neither the a6A nor a6B integrinsubunits. that occurs in response to physiological stimulation does not Stable transfection of either the chick or humana 6 A increase the surface expression of the a6Pl integrin [3, 8]. cDNAs into P388D1 cells resulted in chimeric a6A81 This finding, along with similar observationsmade on develsurface expression
Summary
From the SPromam in Cell and Deueloomentai Biology and Leukocytes use thea681 integrin to adhere to lami- It is becoming apparent that the abilityof cells to interact nin based on mAb inhibition and affinity chromatog- with basement membrane componentsis physiologically regraphy studies This adhesion requires leukocyte stim- ulated [1,2,3,4,5]. Much of the work on regulated laminin adhesion, to date, has focused on therole of the a601 integrin and iesxemplified by studies on leukocyte adhesion What has emergedfrom these studiesis that theability of macrophages [1,2,8], CD4’ T cells [3], and neutrophils [4] to adheret o laminin is markthis mouse macrophage cell line does not adhere to edly enhanced in response to PMA’ or cytokine stimulation.
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