Immunology 1992 77 214-218

Monoclonal antibodies to the murine VLA-6 a-chain trigger homotypic lymphocyte aggregation R. P. WUTHRICH Division of Nephrology, University of Alabama

at

Birmingham, Birmingham, Alabama, U.S.A.

Acceptedfor publication 10 May 1992

SUMMARY Very Late Antigen-6 (VLA-6) is a laminin receptor found on T cells, macrophages and platelets and may function as an activation antigen. Here we describe that two monoclonal antibodies (mAb) targeting the VLA-6 a-chain are capable of inducing homotypic aggregation of murine T-cell lines (3A9 and CIO cells). 3A9 and CIO cells are of the memory phenotype (CD4+, CD8-, CD44+, CD45+) and express VLA-6 abundantly. The VLA-6-induced aggregation is temperature dependent, energy requiring, and involves the cytoskeleton. In addition, divalent cations (Ca2+ and Mg2+) are also necessary for aggregation. The VLA-6-induced aggregation is not inhibitied with mAb targeting the LFA-l/ICAM-l, VLA-4/VCAM-l and CD2/LFA-3 adhesion pairs. We conclude that VLA-6 has a broader function and can serve as an activation molecule, triggering homotypic aggregation of T-cell subsets.

INTRODUCTION Very late activation antigen-6 (VLA-6) is a heterodimeric cell surface glycoprotein expressed by T cells and macrophages and functions as a laminin receptor.'3 The unique a-chain of VLA-6 shares a common /,-chain with other VLA molecules.4 The o6-chain can also associate with a 34-chain .56 The 46/34 complex has a more restricted distribution and is generally not found on cells of haematopoietic origin.7 It has been suggested that the interaction of T cells with laminin via VLA-6 may play a role in T-cell migration and antigen-specific T-cell recognition.8 In addition to known T-cell-activation antigens (such as CD2, CD3, CD28, CD44 and CD45) some of the VLA proteins may also play a role in T-cell activation. VLA-4, VLA-5 (fibronectin receptors) and VLA-6 (laminin receptor) are capable of participating in CD3-mediated T-cell activation8 and VLA-4 can trigger homotypic aggregation of T and B cells when targeted with an a4-specific monoclonal antibody (mAb).9 Here we report for the first time that mAb to VLA-6 can also induce homotypic lymphocyte aggregation. This VLA-6-induced aggregation is independent of LFA- 1 /ICAM- I interactions, and requires energy and an intact cytoskeleton. Thus, in addition to being a matrix receptor, VLA-6 appears to have a broader function and may function as an activation molecule for T cells. MATERIALS AND METHODS Antibodies The following mAb were used in this study (determinant, isotype and source in parentheses): GoH3 (anti-VLA-6a, rat Correspondence: Dr R. P. Wuthrich, Division of Nephrology, University of Alabama at Birmingham, LHR 668B, UAB Station, Birmingham, AL 35294, U.S.A.

IgG2a; A. Sonnenberg, Amsterdam, The Netherlands);'0 13513C (anti-VLA-6a, rat IgG2a; S.J. Kennel, Oak Ridge, TN);" M 17/4.2 [anti-LFA- I a (CD1 a), rat IgG2a; ATCC, Rockville, MD]; M18/2.a.8 (anti-LFA-/l3, rat IgG2a; ATCC); YN1/1.7.4 (anti-ICAM-1, rat IgG2a; ATCC); R1-2 (anti-VLA-4x, rat IgG2b; Pharmingen, San Diego, CA); M/K-2.7 (anti-VCAM- 1, rat IgGI; P. Kincade, Oklahoma City, OK); RM2-5 (anti-CD2, rat IgG2b; Pharmingen); IM7.8.1 (anti-CD44, rat IgG2b; ATCC); M1/89 (anti-CD45, rat IgG2b; ATCC). All antibodies were used as culture supernatants, except for R1-2 and RM2-5 which were used purified. Cell lines The 3A9 T-hybrid cell line'2 was provided by P. Allen (St Louis, MO). C1O and A2A2 T hybrids'3 were kindly provided by L. H. Glimcher (Boston, MA). WEHI-3 and EL-4 cells were obtained from ATCC. All cells were grown in RPMI-1640 supplemented with 10% foetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 pg/ml), HEPES (10 mM), and 2 mM L-glutamine (RPMI-10). Aggregation assays T cells (1 5 x 105/well) were placed onto 96-well plates in 100 Pl RPMI-10. Diluted mAb were then added, and the plates were incubated at 370 in 5% CO2 in a final volume of 150 pi of RPMI10. The plates were then read after 30 min, 1,2,4, 6, 18 and 24 hr and aggregation was scored on a semi-quantitative scale from 0 to 5 + adapted from Rothlein et al.:'4 0, no aggregation; 1 +, 0-20% of the cells are aggregated; 2+, 20-40% aggregation; 3+, 40-60% aggregation; 4+, 60-80% aggregation; 5+, 80100% aggregation. All cell lines, mAb and inhibitors were tested at least three times. Plates were also photographed on an Olympus inverted microscope with phase contrast.

214

215

mAb and murine VLA-6 x-chain Cell ELISA Expression of cell surface determinants was measured by cell ELISA as described previously.'5 Briefly, cells (2 x 106) were washed with cold phosphate-buffered saline (PBS) supplemented with 5% FCS, and were then incubated with primary mAb supernatant at 1: 10. After a 1-hr incubation at 4°, the cells were washed twice with PBS/5%, and were then incubated with horseradish peroxidase-conjugated goat anti-rat Ig at 1:400 for 1 hr. The cells were then washed twice, and were incubated with o-phenylenediamine (50 yg/ml) in 0- 1 M phosphate-citrate buffer (pH 5 0) containing 0-004% H202. The colour reaction was stopped after 8 min by adding 20% v/v concentrated sulphuric acid. The colour in the supernatant was then quantitated by transferring the supernatant to 96-well plates, and reading the OD at 490 nm in an ELISA reader.

RESULTS Two anti-VLA-6a mAb induce homotypic aggregation We have found that the T-hybrid cell lines 3A9 and CO have the ability to aggregate when elicited with the anti-VLA-6cz mAb GoH3 and 135-13C (Fig. 1). The aggregation induced with GoH3 is more potent than the aggregation seen with 135-13C. Aggregation can be detected after only 15-30 min. Maximal aggregation occurs after 6-12 hr. Thereafter cells disaggregate, and only minimal aggregation is seen after 24 hr of incubation with GoH3 or 135-13C. The aggregation is specific for GoH3 and 135-13C, and is not seen with an isotype-matched control antibody targeting the CD18 molecule (mAb M18/2.a.8). Aggregation can also be demonstrated with some other mAb, including anti-LFA- a and anti-CD44, but not with other mAb targeting activation molecules such as CD3 (not shown). Aggregation occurs at extremely low concentrations of GoH3 (Fig. 2). Dilutions of supernatant as low as 1:40,000 still produce visible cell aggregation. Sixty to 80% of the cells are aggregated at dilutions of 1: 100 to 1: 1000 (Fig. 3). Cells can easily be disaggregated with vigorous pipetting. Upon reincubation at 370 the cells reaggregate within 1-3 hr to previous levels.

100

80 a 0

60

0)

0) L-

40

I00

20 0

0

4

8

12

16

20

24

Time (hr)

Figure 1. Time-course of anti-VLA-6a-induced homotypic aggregation in 3A9 cells. 3A9 cells (1 *5 x 105/well) were plated into 96-well plates and were aggregated with GoH3, 135-13C (anti-VLA-6a) or M18/2.a.8 (anti-LFA-lfl) mAb supernatant at 1:1000 (all mAb are IgG2a). Aggregation was read semi-quantitatively at indicated time-points.

100

-

80

-

c 0

60

0)

40

-

0

20

o

o

0T

0

GoH3 dilution Figure 2. Dose response of anti-VLA-6a-induced homotypic aggregation. 3A9 cells (1 5 x 105/well) were plated into 96-well plates, and serial dilutions of GoH3 mAb were added. Aggregation was read semiquantitatively after 12 hr.

Several other T-cell lines

were

screened for their ability to

aggregate in response to GoH3 or 135-13C. Table 1 shows that the aggregation was most intense in 3A9 cells. The VLA-6+ T-hybrid cell line C IO aggregates slower and less intensely when

compared with 3A9 cells. VLA-6+ A2A2 cells (T hybrid), myelomonocytic WEHI-3 and VLA-6- EL-4 T cells do not aggregate in response to GoH3. Also normal splenocytes do not aggregate in response to GoH3. Physiological requirements of anti-VLA-6-mediated cell aggregation Table 2 summarizes the physiological requirements of cell aggregation in response to GoH3. The aggregation of 3A9 is temperature dependent, and no aggregation occurs at 4°. Upon warming of the cultures back to 370, prompt reaggregation occurs at a normal rate within 1-3 hr to previous levels. Pretreatment of the cells for 30 min with a mixture of the metabolic inhibitors 2-deoxyglucose (5 mM) and sodium azide (0 1 %) effectively blocks the aggregation process. Pretreatment of 3A9 cells with the cytoskeletal inhibitor cytochalasin B (10 pM) also completely blocks aggregation (Fig. 3d). Colchicine (10 gM), an inhibitor of microtubules has no effect. These experiments suggest that the VLA-6a-induced homotypic aggregation process requires an active metabolism, an intact cytoskeleton, and that it is a physiological event and not simply Ig-mediated agglutination. When the aggregation is performed in Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS) rather than in RPMI-10, no aggregation is seen. The aggregation in Ca2+ and Mg2+containing RPMI- 1640 media without FCS is suboptimal, indicating that in addition to Ca2+ and Mg2+ other serum factors are required for aggregation. Pretreatment of cells with the divalent cation chelator EDTA (1-5 mM) blocks the aggregation in response to GoH3 (Fig. 4). The calcium chelator EGTA (1-5 mM) is less effective, but also inhibits the aggregation, and accelerates the disaggregation process. Inhibition of homotypic aggregation with mAb Inhibition studies with mAb were then performed to determine potential ligands which might be involved in mediating the

216

R. P. Wuthrich

Figure 3. Photomicrographs of anti-VLA-6a-induced homotypic aggregation in 3A9 cells (original magnification x 200). (a) Control cells incubated without mAb, demonstrating very weak baseline aggregation (1 +). (b) Cells incubated with anti-VLA-6a mAb GoH3 at 1:100 (4+ aggregation). (c) Cells incubated with GoH3 at 1:1000 (3+). (d) Cells were preincubated with cytochalasin B (10 pM) and were then elicited with GoH3 (1: 100) (0 aggregation). (e) Cells were pretreated with anti-LFA-1I mAb M 18/2.a.8 (1: 10), and were then co-incubated with GoH3 (1:100) (4+ aggregation).

Table 2. Physiological requirements for anti-VLA-6cx-mediated

aggregation

Table 1. Homotypic aggregation in response to anti-VLA-6a mAb GoH3

Condition VLA-6 Cell line

Phenotype

expression

Homotypic aggregation

3A9 CIO A2A2

CD4+CD8CD4+CD8CD4+CD8-

EL-4 WEHI-3

CD4+CD8CD4-CD8-

-

0 0

Splenocytes

CD4+CD8-

+

0

+ + + -

4+ 2+ 0

Various murine T-lineage cells and splenocytes were aggregated with anti-VLA-6 mAb GoH3 at 1: 100. Aggregation was determined after 12 hr (24 hr for C 10 cells), and was scored on a semi-quantitative scale from 0 to 5 +. VLA-6o cell surface expression was measured by cell ELISA.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Aggregation

370

4+

40

0

40

then 370 2-deoxyglucose (5 mM) + sodium azide (0- 1 %) Cytochalasin B (10 pM) Colchicine (10 pM) Ca2+- and Mg2+-free HBSS RPMI-1640 EGTA (5 mM) EDTA (5 mM)

4+ 0 0

4+ 0

2+ 2+

1+

3A9 cells were aggregated with GoH3 (1:100) for 6 hr under the above conditions, and were scored from 0 to 5 +. Conditions 1-6 and 910 were performed in RPMI-10 (containing 10% FCS).

217

mAb and murine VLA-6 ax-chain 100

80 GoH3 control EGTA EDTA

60

+ +

40

20 0

2

0

4

6

GoH3-induced aggregation. Table 3 shows that of the examined mAb, none had the ability to block the aggregation process. Over the 12-hr observation period no significant inhibition could be demonstrated with any of the tested mAb. In particular, targeting the LFA-l/ICAM- 1, VLA-4/VCAM-1 and CD2/LFA-3 adhesion pairs had no inhibitory effect on homotypic aggregation in 3A9 cells. All these mAb were tested on various cell lines by flow cytometry and/or immunoprecipitation for positive staining to ascertain specificity of the antibody preparation. Furthermore, these antibodies were functional in inhibiting adhesion of leucocyte cell lines to epithelial cells (not shown).

Time (hr)

Figure 4. Inhibitory effect of EGTA (1 mM) and EDTA (1 mM). 3A9 cells aggregated with GoH3 at 1: 100 in the presence of these chelators and aggregation was scored semi-quantitatively. were

Table 3. Effect of inhibitory mAb on GoH3-mediated homotypic aggregation

Stimulatory mAb GoH3(1:100) GoH3 (1: 100) GoH3 (1:100) GoH3 (1:100) GoH3 (1:100) GoH3 (1:100)

Inhibitory mAb

YN1/1.7.4 M 18/2.a.8 M/K-2.7 Rl-2 RM2-5

Aggregation

1+ 4+ 4+

(anti-ICAM-1; 1: 10) (anti-LFA- l ; 1: 1 0) (anti-VCAM-1; 1: 10) (anti-VLA-4o; 10 pg/ml) (anti-CD2; 10 pg/ml)

4+ 4+ 4+ 4+

3A9 cells were stimulated for 12 hr with GoH3 mAb in the presence of the indicated mAb targeting various adhesion molecules. Aggregation was scored semi-quantitatively on a scale from 0 to 5.

control

VLA-6 ICAM-1 LFA-1 VCAM-1 IzzZ/zz VLA-4 CD2 IzzX/A a CD44 I CD45 0.0

0-2

Phenotype of 3A9 cells To examine the cell surface characteristics and to correlate them with the aggregation results we measured the expression of several T-cell markers by cell ELISA, using a panel of mAb. Figure 5 demonstrates that 3A9 are CD4+, CD8-, CD44+ and CD45+ T cells (T-memory cell phenotype). 3A9 cells express LFA- 1, but not ICAM- 1, and are negative for VLA-4/VCAM- I and CD2. 3A9 are positive for VLA-6a, but negative for the VLA-4a chain. Thus, since ICAM-1, VLA-4 and CD2 are not expressed on these cells the aggregation appears to be mediated by other cell surface proteins.

M%~

I

I

I

0-4

0-6

0-8

OD (490 nm)/million cells

Figure 5. Cell surface expression of various adhesion molecules and integrins on 3A9 cells. Cells (2 x 106/sample) were stained by cell ELISA as indicated in Materials and Methods. Results represent OD/106 cells. Control represents background staining obtained when incubating cells with secondary antibody only.

DISCUSSION The #,-integrin VLA-6 has primarily been recognized as a laminin receptor. VLA-6 binds the E8 fragment of laminin in an RGD-independent fashion.2'3 Thus far other functions for VLA-6 have not been found, but have been demonstrated for a related molecule, the VLA-4 (x4j,-integrin). Initially it was shown that VLA-4 was a fibronectin receptor. Later it was found that VLA-4 also binds to Vascular Cell Adhesion Molecule-i (VCAM-l ).16 VLA-4 has yet another function and can trigger homotypic aggregation in human T- and B-cell lines.9 Here we show that VLA-6 can also induce homotypic cell aggregation of lymphocytes, suggesting that this VLA integrin also has additional functions. The anti VLA-6 mAb GoH3 and 135-1 3C are very powerful antibodies and are capable of inducing aggregation at extremely low concentrations (in the picomolar range). This suggests that VLA-6 might be a signalling molecule which programmes the cells to aggregate. The homotypic aggregation process requires an intact cytoskeleton and metabolic energy. These observations are reminiscent of the findings reported for VLA-4- and Thy-1-induced homotypic aggregation of immune cells which require similar physiological conditions.9"'7 VLA-6 is known to be associated with the cytoskeleton,'8 and may thus mediate interaction between matrix (and perhaps adhesion molecules) and the cytoskeleton. Our studies show that mAb targeting the LFA- I/ICAM-1, VLA-4/VCAM-1 and CD2/LFA-3 adhesion molecules fail to inhibit the homotypic aggregation induced with anti-VLA-6 mAb GoH3. Furthermore, 3A9 cells do not express significant levels of ICAM-l, VLA-4, VCAM-I or CD2. This suggests that other adhesion molecules mediate the binding of these cells when elicited with GoH3. It is not clear from our studies which other molecules are mediating the aggregation. VLA-6 might bind to

a

yet unknown adhesion molecule found

on

3A9 cells.

R. P. Wuthrich

218

Another possibility is that VLA-6 is a regulatory molecule that influences some other adhesive mechanism. Precedent for a surface protein regulating other adhesion proteins has been reported for several other molecules, including CD 14,19 CD4020 and CD43.2' Targeting these proteins with monoclonal antibodies induces homotypic aggregation which can be blocked by anti-LFA-I mAb. In summary, we have identified two murine T-cell lines that have the ability to display homotypic aggregation when elicited with anti-VLA-6a mAb. This aggregation process is dependent on divalent cations, metabolic energy and requires an intact cytoskeleton. The VLA-6 triggered aggregation does not involve ICAM-1/LFA-1, VCAM-l/VLA-4 or CD2/LFA-3 binding, but may be mediated by yet unknown adhesion molecules.

9. 10.

11. 12.

13.

ACKNOWLEDGMENTS I would like to thank P. Sekar for technical assistance and Dr D. G. Warnock for his support and encouragement.

14.

REFERENCES

15.

1. MECHAM R.P. (1991) Receptors for laminin FASEB J. 5,2538.

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2. HALL D.E., REICHARDT L.F., CROWLEY E., HOLLEY B., MOEZzI H., SONNENBERG A. & DAMSKY C.H. (1990) The aI/# I and a6/f3I integrin heterodimers mediate cell attachment to distinct sites on laminin. J. Cell Biol. 110, 2175. 3. SONNENBERG A., LINDERS C.J.T., MODDERMAN P.W., DAMSKY C.H., AUMAILLEY M. & TIMPL R. (1990) Integrin recognition of different cell-binding fragments of laminin (PI, E3, E8) and evidence that a6/fl but not a6/?4 functions as a major receptor for fragment E8. J. Cell Biol. 110, 2145. 4. HEMLER M.E. (1990) VLA proteins in the integrin family. Ann. Rev. Immunol. 8, 365. 5. KAJIJI S., TAMURA R.N. & QUARANTA V. (1989) A novel integrin (a6/fl4) from human epithelial cells suggests a fourth family of integrin adhesion receptors. EMBO J. 8, 673. 6. KENNEL S.J., FooTE L.J., FALCIONI R., SONNENBERG A., STRINGER C.D., CROUSE C. & HEMLER M.E. (1989) Analysis of the tumorassociated antigen TSP-180. Identity with 26/f4 in the integrin superfamily. J. biol. Chem. 264,15515. 7. SONNENBERG A., LINDERS C.J.T., DAAMS J.H. & KENNEL S.J. (1990) The a6/fii (VLA-6) and a6/f4 protein complexes: tissue distribution and biochemical properties. J. Cell Sci. 96, 207. 8. SHIMIZU Y., VAN SEVENTER G.A., HORGAN K.J. & SHAW S. (1990) Costimulation of proliferative responses of resting CD4+ T cells by

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Monoclonal antibodies to the murine VLA-6 alpha-chain trigger homotypic lymphocyte aggregation.

Very Late Antigen-6 (VLA-6) is a laminin receptor found on T cells, macrophages and platelets and may function as an activation antigen. Here we descr...
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