Integrin α₁β₁ (VLA-1) mediates adhesion of activated intraepithelial lymphocytes to collagen

INTRODUCTION

Human jejunal intraepithelial lymphocytes (IELs) are predominantly CD8⁺ T cells situated at the basolateral surfaces of epithelial cells in the intestine. The epithelial layer overlies a basement membrane composed of a matrix of extracellular proteins, particularly collagen, laminin, fibronectin and heparan sulphate proteoglycans.¹ IELs are memory lymphocytes that have homed to the epithelium; they express markers of chronic activation, such as HML-1, CD45RO, and integrin α₁β₁ (very late activation antigen-1; VLA-1).^2–7 Their function, while not completely understood, is likely to include cytotoxic activity against malignant and virally infected epithelial cells.⁸ IELs proliferate minimally and produce little interleukin-2 (IL-2) or interferon-γ (IFN-γ), except when stimulated through the CD2 receptor.^9,10

Several modes of IEL adhesion occur in the epithelium. IELs bind epithelial cells through the HML-1/E-cadherin and lymphocyte function-associated antigen type-1 (LFA-1)/intracellular adhesion molecule-1 (ICAM-1) receptor pairs.^11,12 Similarly, adhesion to mesenchymal cells that underlie the basement membrane, such as smooth muscle cells and fibroblasts, is mediated by VLA-4 and LFA-1.⁴ A third component of IEL anchoring in the epithelium may be their binding to extracellular matrix (ECM) proteins, the subject of the present study.

The types of mesenchymal cells and ECM proteins that occur in various tissues are site-specific. For example, colonic and dermal fibroblasts produce different types of collagen.¹³ Even within the intestinal mucosa, the collagen type varies among sites, with type IV produced by subepithelial fibroblasts and type V found mainly in the submucosa.^14,15

The VLA integrins, membrane proteins composed of α-chains 1 to 6 paired with the β₁-chain, serve as receptors for ECM proteins and mesenchymal cells. Specifically, VLA-1, VLA-2 and VLA-3 are collagen receptors. The predominant expression of VLA-1, rather than VLA-2 or VLA-3, by intestinal IELs suggests that the former serves as the collagen receptor for this compartment of lymphocytes. The purpose of this study was to determine what ECM proteins are bound by IELs and the mechanism involved.

MATERIALS AND METHODS

RESULTS

The ability of IL-2-activated IELs to adhere to ECM proteins coated onto culture wells was compared to the same activities of CD8⁺ T cells from peripheral blood. After 1 hr, a large fraction of IELs bound to collagen types I, II and IV (20–31%), whereas fewer bound fibronectin, laminin, or a BSA control. (In the absence of IL-2-stimulation, few IELs bound at all.) In contrast, only 9% and 4% of peripheral blood CD8⁺ T cells bound collagen I and IV, respectively, whereas a large number (32%) bound fibronectin (Fig. 1). When comparing the two lymphocyte types, more IELs bound collagen IV, whereas more peripheral blood CD8⁺ T cells bound fibronectin (both P < 0·05). Fibronectin binding by both lymphocyte types was reduced to BSA control values by adding antibody to VLA-4. The number of adherent IELs reached a maximum by 1–1·5 hr for all ECM proteins tested (not shown), indicating that detected differences in IEL binding were not due to different rates of adhesion. When combined in the same well, pairs of ECM proteins resulted in no greater binding than the more efficacious protein alone (not shown).

Since the activation of protein kinase C enhances many binding events, its effect on IEL adhesion was determined. Fresh IELs were incubated for 10 min with the protein kinase C-activator PMA (100 ng/ml), or the inhibitor staurosporine (1·0 μm), then washed and assayed for adhesion. PMA activation of protein kinase C caused increased binding to collagen types I and IV, but not to fibronectin or laminin. Conversely, staurosporine inhibition of protein kinase C reduced IEL binding whether or not PMA was included in the pretreatment, and, again, occurred only in wells coated with collagen I or IV, not fibronectin or laminin (Fig. 2a). Inhibition of tyrosine kinases by herbimycin (1·0 μm), in contrast, did not alter IEL binding to the four ECM proteins (not shown). Thus, the mechanism of IEL adhesion to collagen requires protein phosphorylation by protein kinase C, not the tyrosine kinases. The effects of receptor-mediated T-cell stimulation were also tested on fresh IELs: a 24-hr incubation with the mitogenic anti-CD2 mAb pair, Tll₂ and Tll₃, caused a larger fraction of IELs to bind collagen (but not fibronectin or laminin), whereas anti-CD3 mAb or PHA had no effect (Fig. 2b).

The role of the VLA integrins in IEL adhesion to ECM proteins was examined next. Immediately before assaying adhesion of IL-2-activated IELs and peripheral blood CD8⁺ T cells, these surface receptors were measured by detection of their respective α-chains using immunofluorescence and flow cytometry. The stimulation of lymphocytes was the same as that used for binding experiments, so the two results may be compared. The majority of IELs expressed α₁ and α₄, fewer expressed α₃ and α₅, while only a small minority carried α₂ and α₆. In comparison, the majority of peripheral blood CD8⁺ T cells were positive for α₃, α₄ and α₅, but not α₁ (Table 1). Both IELs and peripheral blood T cells in two experiments displayed similar levels of each marker before and after IL-2 stimulation (not shown), demonstrating that the VLA integrin is unchanged by IL-2-stimulation. To determine whether the VLA integrins mediate IEL adhesion, blocking mAb were added to the assay medium and the effect was measured on IEL adhesion to collagen types I and IV. Adhesion to both collagen types was partially reduced by mAb against the α₁ chain of VLA-1, but was unaffected by mAbs against α₂ to α₆ (Fig. 3). Blocking the common β₁-chain alone reduced IEL binding significantly, and the combination of anti-α₁ and anti-β₁ almost eliminated binding to both collagen types I and IV, by 82±6% and 94±1%, respectively (P < 0·001), thus implicating VLA-1 as a crucial collagen receptor on IELs (Fig. 3). Taken together, these results show that IELs bind avidly to collagen through their VLA-1 receptors, which depend on protein phosphorylation by protein kinase C.

Receptor-mediated cell adhesion often depends on extracellular divalent cations. In other systems, Ca²⁺ is inhibitory while Mn²⁺ increases binding ^16–18. To determine whether IEL adhesion is subject to the influences of these cations, binding to collagen types I and IV was assayed in the presence of a range of concentrations of MnCl₂, MgCl₂ and CaCl₂. Mn²⁺ caused striking dose-dependent increases in IEL adhesion at concentrations up to 32 mm. Mg²⁺ had similar but less pronounced effects, while Ca²⁺ did not support binding at all (Fig. 4). There was no cell binding in the absence of these cations. Thus, cell adhesion was dependent on Mn²⁺ or Mg²⁺, but not Ca²⁺.

Potential cation interactions were explored by adding two different cation species concurrently at various concentrations and measuring the effect on adhesion to collagen type IV. When Ca²⁺ was combined with Mg²⁺, Ca²⁺ concentrations above 1·0 mm caused dose-dependent reductions in Mg²⁺-supported cell binding. Ca²⁺ at 100 mm reduced adhesion to background levels, regardless of the Mg²⁺ concentration. This is demonstrated by Ca²⁺-response curves that are initially parallel but begin to converge above 1·0 mm Ca²⁺ (Fig. 5a). Similarly, Mn²⁺-supported adhesion was down-regulated by Ca²⁺, an effect dependent on the relative concentration of each cation: at higher Mn²⁺ concentrations, more Ca²⁺ was required to initiate a down-turn in the binding curve and to inhibit to baseline levels, resulting in the parallel curves of Fig. 5(b). At all Mn²⁺ concentrations tested, half-maximal binding occurred when the Ca²⁺ dose exceeded the Mn²⁺ concentration by about 10-fold, indicating competition between these two cations. Finally, combining Mg²⁺ and Mn²⁺ resulted in additive effects on cell adhesion, although Mg²⁺ supported no additional adhesion when the Mn²⁺ concentration exceeded 0·1 mm (Fig. 6). Similar results for all three paired-cation combinations were obtained using type I collagen (not shown).

DISCUSSION

Cell binding to collagen type I is normally mediated by integrins VLA-1, -2 and -3 and some non-integrin receptors, while binding to collagen type IV is mediated by VLA-1 and VLA-2. VLA-2-mediated binding is Mg²⁺-dependent and is inhibited by Ca²⁺. It is implicated in the adhesion to collagen type I by circulating leucocytes, platelets, smooth muscle cells, tumour cells and fibroblasts.^19–22 [VLA-2 is also a receptor for echovirus 1, although unlike VLA-2/ECM binding, this interaction is not inhibited by Ca²⁺, nor stimulated by phorbol esters.²³] Cell binding to collagen type IV utilizing VLA-1 has been shown only with transfectants, liposomes, isolated integrins, and two natural cell types, the smooth muscle cell and the neuroblastoma cell.^24–28 Lymphocytes may act very differently.

The integrin VLA-1 is found on smooth muscle cells, chronically activated T cells and intestinal lymphocytes. Circulating T cells do not express VLA-1 until they have been activated for several weeks with antigen, mitogen, or IL-2 in vitro,³ after which uniform expression is found on CD4⁺ and CD8⁺ T cells. That mucosal lymphocytes express VLA-1 is consistent with the theory that they are chronically activated and have homed to the mucosa. Although IELs are memory cells, their actions are very different from memory cells in the periphery.⁶ In the mucosal epithelium, they are anchored by binding to components of the extracellular environment, including the ECM proteins. Of several ECM proteins tested, only the collagens promoted significant IEL adhesion. Collagen type IV exists in the basement membrane, and its interaction with VLA-1-expressing T cells may be specific for mucosal sites.

The present report demonstrates that VLA-1 is the main receptor to mediate adhesion to collagen by IL-2-stimulated IELs, in vitro, unlike other leucocytes which utilize VLA-2 and VLA-3. Any role of the latter two in IEL adhesion is undetectably small, as few IELs expressed α₂ or α₃, and mAb against them did not inhibit binding. VLA-1 did not mediate IEL adhesion to laminin; the few cells that bound this substrate were not inhibited by antibody against α₁. While a previous study implicated VLA-1 in the adhesion of smooth muscle cells to both collagen and laminin, these cells are very different from IELs and may express other laminin-binding integrins.

The percentages of IL-2-activated peripheral blood T cells that bound fibronectin were 15% and 30% in two reports.^29,30 The present study shows a comparable 32% binding by peripheral blood CD8⁺ T cells. IEL binding to fibronectin was 11% with PMA stimulation and 10% with IL-2, suggesting that the stimulus is not an important variable. The low IEL binding may be due to their CD8⁺ phenotype, since CD4⁺ T cells have been shown to adhere better to fibronectin.^29,30 Although memory cells adhere to fibronectin more avidly than do naive cells, IELs (also memory cells) differ considerably from those in the peripheral blood.⁶ Thus, low fibronectin binding by IELs cannot be completely explained at this time.

Protein kinase C frequently regulates leucocyte adhesion. It probably promotes conformational changes in surface integrin molecules by stimulating their interaction with cytoskeletal elements. The activation of protein kinase C is shown here to be a prerequisite for collagen adhesion by IELs, as staurosporine inhibited and PMA enhanced cell binding. Also, the increased cell adhesion that occurred upon the addition of anti-CD2 mAbs, but not anti-CD3 mAb or PHA, may be due to greater protein kinase C activation. (Of these stimuli, CD2 ligation also triggers the most proliferation by IELs, another response that depends on protein kinase C activity). This also shows that the low proliferation of IELs in response to CD3 ligation or PHA is probably due to a defect proximal to protein kinase C activation.

The adhesion of IELs to collagen was found to require extracellular divalent cations, a characteristic demonstrated previously with other integrin-dependent cell binding events, including VLA-1-mediated lymphocyte adhesion.²⁴ IEL adhesion to collagen was better supported by Mn²⁺ than by Mg²⁺. Both effects were inhibited by Ca²⁺, but in different ways that give clues to how the ions bind to the receptor molecule to influence its affinity for collagen.

The extent to which Mn²⁺-supported adhesion was inhibited by Ca²⁺ depended on which ion predominated in the medium. Thus, in the absence of Ca²⁺, Mn²⁺ may bind to the receptor enabling it to bind collagen, while bound Ca²⁺ disables the receptor. In the presence of both cationic species, the one occurring at the higher concentration relative to its dissociation constant prevails. In the absence of both ions, the unoccupied ion-binding site(s) makes the receptor non-functional (but subject to the influence of Mg²⁺). These effects may be due to a common binding site in the receptor molecule for which Ca²⁺ and Mn²⁺ compete directly or to two distinct ion-binding sites where competition occurs indirectly.

In comparison, the ability of Ca²⁺ to inhibit Mg²⁺-dependent adhesion was independent of the Mg²⁺ concentration. This must require another ion-binding site for Mg²⁺ exclusively, the occupation of which turns on the receptor. Rather than Ca²⁺ competing for this second ion binding site, the effectiveness of Mg²⁺ in enabling the VLA-1 receptor seems to be modulated by the Ca^2+/Mn²⁺ binding site(s), since Mg²⁺-supported cell adhesion is inhibited by Ca²⁺ and enhanced by Mn²⁺. In this way, occupation of the ion-binding sites by divalent cations in the extracellular fluid would determine the functional state of the VLA-1 receptor: enabled to bind collagen with Mg²⁺ or Mn²⁺ bound; or non-functional with Ca⁺⁺ bound or all sites empty. It is likely that ion-binding induces conformational alterations in the VLA-1 receptor that affect its affinity for collagen. Further elucidation of these effects and confirmation of the model await molecular analysis.

The function of VLA-1-mediated adhesion of IELs to collagen may be for homing and anchorage. The interaction of cellular receptors with ECM proteins has been shown to trigger many events, such as collagen matrix contraction, cell activation and migration. In the mucosa, collagen type IV of the basement membrane may anchor IEL that have migrated into the epithelium from the lamina propria in response to IL-8, which is secreted by epithelial ³¹ and attracts IELs.³² Once in the epithelium, IELs are anchored by several chronic activation antigens that are absent from most circulating lymphocytes: HML-1 binds epithelial cells, VLA-4 binds mesenchymal cells and VLA-1 mediates collagen adhesion. Like IELs, intestinal epithelial cells bind collagen IV more than the other ECM proteins.³³ The concentrations of the divalent cations may differ in the intestine, such as the high concentration of Mn²⁺ in bile, which bathes the jejunum. In wounds, Mg²⁺ concentrations increase while Ca²⁺ concentrations decline, changes that would lead to increased adhesion.³⁴