CD44 as an hyaluronic acid receptor on T cells
Eur. J. Immunol. 1992. 22: 2719-2723
Jayne Lesley and Robert Hyman Department of Cancer Biology, The Salk Institute, San Diego
2719
CD44 can be activated to function as an hyaluronic acid receptor in normal murine T cells* The hyaluronic acid (HA)-binding function of CD44 expressed on the cell surface of normal hematopoietic cells has been studied by assaying binding of fluoresceinated hyaluronic acid (FI-HA) and adhesion to immobilized HA. As has been observed previously, normal hematopoietic cells from bone marrow and spleen do not constitutively bind HA. A CD44-specific monoclonal antibody, IRAWB 14, which has been shown to rapidly induce H A binding in some CD44+ cell lines,was used to activate the HA-binding function of CD44 in these normal cells. Only splenic T cells were activated by the IRAWB 14 antibody to bind F1-HA. Upon activation, FI-HA binding correlated with the level of CD44 expression. Activation of H A binding allowed splenicT cells to adhere to H A immobilized on plastic and to an endothelial cell line in an HA-dependent manner. BALBlc and AKR/J splenicT cells differ in their level of CD44 expression, and this correlated with differences in their ability to bind H A upon antibody activation. The minor subpopulation of MEL-14- T cells were among the brightest F1-HA-staining cells. We propose, on the basis of these and other results, that there are three states of CD44 function with respect to H A binding: (a) a non-activatable, resting state, which cannot be rapidly activated to bind H A , as seen in most hematopoietic cells; (b) an activatable state, which can be rapidly converted to HA-binding function, in this case by the IRAWB 14 antibody, illustrated by Tcells as shown here; and (c) a constitutively active state, which can bind H A without antibody activation, seen in some cell lines.
1 Introduction In vitvo, CD44 has been shown to be a receptor for hyaluronic acid (HA), a high-molecular weight, repeating disaccharide, that is present in extracellular matrix and body fluids [ 1-51. However, most normal hematopoietic cells of the mouse which express CD44 do not constitutively bind H A in vitro [3, 5 , 61. We have described a CD44specific mAb, IRAWB 14, which rapidly “activates” HAbinding function in some CD44+, HA-receptor- cell lines [5].We use the tcrm “activation” to indicate the conversion of pre-existing CD44 molecules from a non-HA-binding state to a form able to bind HA.This is distinguished from the effects of phorbol esters on some T lymphomas, which occur over several hours and are accompanied by increased CD44 expression [3, 71. Here, we show that the CD44specific mAb, IRAWB 14, also activates HA-receptor function in normal murine T cells that express CD44. Differences in the level of CD44 expressed on T cells of BALB/c mice and AKR/J mice correlate with the HA-
binding capacity of these cells. Splenic B cells and bone marrow cells are not activated t o bind H A by the IRAWB 14 antibody.
2 Materials and methods 2.1 Cell preparation
Single cell suspensions of spleen and bone marrow were prepared from AKR/J and BALB/c J mice (Jackson Laboratories, Bar Harbor, ME).T cells were enriched from spleen by panning on petri dishes coated with antibody t o immunoglobulin. After panning 650/0-850/0 of the recovered nonadherent cells were shown to beT cells on the basis of staining with fluoresceinated antibodies against Thy-1 alleles. B cells were enriched from spleen by two rounds of cytotoxic depletion with cytotoxic Thy-1-specific antibodies and rabbit complement, and recovery of live cells by centrifugation over Lymphoprep (Nycomed AS, Oslo, Norway) [S]. These populations contained no cells staining with fluoresceinated anti-Thy-1 antibodies. The murine LN endothelial cell line, TME-3H3 [5, 91, was harvested with trypsin and replated at 1 x lo4 -2 x 104/wellon 24-well [I 106251 plates 1 or 2 days before use in adhcsion assays.
* This work was wpportcd by National Cancer Institute Grant CA-13287 and Natmnal Institute of Allergy and Infectious Diseaxs Grant AT-31613 to R. Hyman, by National Cancer Institute Core Grant CA-14195 to the Salk Institute and by thc Hansen Foundation. Correspondence: Jayne Lesley, Department of Cancer Biology, The Salk Institutc, PO. Box 85800, San Diego, CA 92186-5800, USA Abbreviations: HA: Hyaluronic acid FI-HA: Fluorescein-conjugated hyaluronic acid
0 VCH Vcrlagsgesellschaft mbH, D-6940 Weinheim, 1992
2.2 Flow cytometry
Cell surface antigen expression was determined by flow cytometry using a FACScan (for one- and two-color analysis) or FACStar+ (for three-color analysis) (Becton Dickinson, Mountain View, CA). Antibodies and cell staining are as described in [lo] and [ 111, and in the Figure legends. Coumarin-labeled 30H12 was used for three-color analysis [ll]of BALB/c spleenT cells, to allow gating o n T 0014-2980/92/1010-2719$3.50+ .25/0
2720
J. Leslcy and R. Hyman
cells, and staining with FI-HA and biotinylated antibody plus PE-streptavidin. Fluorescein-conjugated hyaluronic acid (Fl-HA) was prepared and used as previously described [3]. Activation of Fl-HA binding was achieved by simultaneous addition of S pg/ml of purified IRAWB 14 mAb [5]. Specificity of Fl-HA binding was confirmed by blocking with an excess of unlabeled H A (100 pg/ml final concentration).
2.3 Adhesion assays Adhesion assays using T-enriched spleen cells were performed as previously described [5] using 24-well plastic plates coated with HA or bearing monolayers of the TME-3H3 lymph node endothelial line [9]. mAb (as a 1: 20 dilution of hybridoma culture supernatant) were added to Tr-labeled, T-enriched spleen cells just prior to their addition to washed, substrate-coated plates. Percent adhesion was calculated as: cpm adhering + (input cpm spontaneously released cpm). HA dependence of adhesion to TME-3H3 cells was determined by hyaluronidase treatment of TME-3H3 monolayers [ S ] .
Eur. J. Immunol. 1992. 22: 2719-2723 The mean fluorescence of the induced population was only about two-fold greater than the mean of the same population in the presence of unlabeled HA. In an effort to identify other agents capable of rapidly inducing HA-binding activity in T cells, in a manner that would mimic the effect of TRAWB 14 antibody, AKR/J and BALB/c splenic T cells were incubated at 37 "C for 20 min with the following reagents: the phorbol ester PMA, the Ca2+ ionophore ionomycin, PMA ionomycin, Con A, PMA-induced EL4 cell supernatant as a source of IL-2, CD3-specific antibody cross-linked with antibody to hamster immunoglobulin, IL-3, IL-4 and IL-7. FI-HA was then added for 15 min at 4 "C and the cells were subsequently washed and examined by flow cytometry. None of these treatments affected Fl-HA binding by the treated T cells. Culture of splenic T cells overnight in PMA also did not induce binding of F1-HA ([3] and data not shown).
+
FL-anti-CD44
FL-HA alone
FL-HA+IRAWB14
3 Results 3.1 Normal murine T cells, but not other CD44+ hematopoietic cells, can be induced to bind Fl-HA Cell populations prepared from spleen and bone marrow of AKR/J and BALB/c mice were stained with fluoresceinconjugated TM7 mAb (to detect CD44 expression) and with Fl-HA and analyzed by flow cytometry (Fig. 1). These mouse strains represent two extremes in the level of CD44 expression on thymocytes and T cells [lo, 121. Column 1in Fig. 1 illustrates this difference in expression of CD44 on peripheral T cells.While AKR/J Tspleen contains a CD44low or -negative population, a CD44-intermediate population and a CD44-high population, BALBkTspleen cells all express relatively high levels of CD44. B cells in both BALBk and AKR/J mice express uniformly high levels of CD44, but AKR/J bone marrow contains a CD44-negative population not present in BALB/c bone marrow. When the binding of Fl-HA by these populations is examined (Fig. 1, center column), using blocking by unlabeled H A (dotted curve) as the criterion for specificity, no specific HA binding by any of the populations can be demonstrated. However, in the presence of IRAWB 14 antibody, a distinct population of F1-HA binding T cells is seen (Fig. 1, last column, first and second panels). This binding is competed by unlabeled H A (dotted insert). In spleen from AKR/J mice, the F1-HA-positive population only contains about one third of the T cells, corresponding to the brightest CD44-staining population (see below), while in BALB/c spleen, most T cells are positive at some level. Splenic B cell-enriched populations and total bone marrow in both mouse stains showed a slight positive shift in Fl-HA staining in the presence of IRAWB 14,which was competed by unlabeled H A (Fig. 1, panels 3-6 in the last column).
I
B Cells
Balb/c Splenic B Cells
!/;,..A1 I
1
Relative Fluorescence Intensity
Figure 1. Expression and HA binding function of normal hematopoietic cells: T-enriched spleen cells (rows 1 and 2), B-enriched spleen cells (rows 3 and 4), and total bone marrow (rows 5 and 6). In the first column, CD44 expression was determined using FITC-conjugated CD44-specific antibody IM7. Dotted insert is the background fluorescence of unstained cells. In the second column, cells were stained with FI-HA.The dotted insert is F1-HA staining in the presence of excess unlabeled HA. In the third column, cells were stained with FI-HA in the presence of 5 Kgglml of purified IRAWB 14 antibody. The dotted insert is FI-HA staining in the presence of IRAWB 14 and excess unlabeled HA.
3.2 Hyaluronic acid-dependent adhesion can be activated in T cells AKR/J T-enriched spleen cells were used to study induction of cell adhesion to hyaluronic acid. Cells were treated with CD44-specific mAb KM201, which inhibits CD44-dependent binding of H A [l], or with CD44-specific mAb IRAWB 14, which activates CD44-dependent binding of F1-HA, and the treated cells were assayed for adhesion to plastic wells coated with H A or to the endothelial cell line TME-3H3 (Table 1). There was very little adhesion to HA-coated plates without induction. IRAWB 14 mAb activated significant adhesion to HA-coated culture wells and caused increased adhesion to endothelial cells. As observed previously with cell lines [5], a portion of the binding of untreated cells to endothelial cells was not CD44 dependent, since it could not be inhibited by KM201 mAb. Hyaluronidase treatment of TME-3H3 cells abolished the increase in T cell adhesion that was induced by IRAWB 14 an ti body (not shown) .
3.3 F’l-HA binding correlates directly with CD44 expression In two-color flow cytometric analysis of splenic T cells, F1-HA binding cells (induced in the presence of IRAWB 14 mAb) were also stained with biotin-labeled antibodies against other cell surface antigens plus PE-labeled streptavidin. In both AKR/J spleen and BALB/c spleen, the intensity of F1-HA staining was directly proportional to the level of CD44 expression (Fig. 2 A, B). T cells were specifically identified by the expression of TcR (Fig. 2 C, D), CD4 or CD8. A proportion of CD4+ and CD8+ cells were positive for FI-HA in both strains. However, in BALB/c spleen, the majority of CD4+ cells were positive for Fl-HA staining, while in AKR/J spleen most of the CD4+ cells did not bind F1-HA or bound very weakly (data not shown). The difference in Fl-HA staining among CD4+ cells accounts for much of the difference in F1-HA binding in BALB/c splenic T cells versus AKR/J splenic T cells.
103
8
g
102
0
?
s
10’
W
a 100
TCR
None
KM201b) IRAWB 14
YOadhesion to HA plates
% adhesion to 3H3 cells
6.9% k 0.1 2.8% k 0.3 34.0% k 7.8
25.4% k 4.0 14.5% k 3.1 44.5% k 2.2
a) Mean and standard error of four replicate samples. b) KM201 i s a CD44-specific mAb that has been shown to inhibit CD44 binding to H A [I]. Binding in the presence of KM201 antibody (not inhibited by KM201) is considered to be nonCD44 dependent (ie., nonspecific, or due to specific factors other than CD44).
D
C
103
I
io1
10‘
103
. . . . .J...? : 101
100
,....,.....,......
102
103
FI-HA fluorescence
Figure 2. Relationship of IRAWB 14-induced F1-HA binding to expression of CD44 and Tcell receptor. F1-HA binding was induced on splenic T cells by treatment with mAb IRAWB 14. CD44 was detected with biotinylated-IM7 antibody (A and B) and TcR was detected with biotinylated -57.597 (specific for afi TcR; C and D). Biotin-antibodies were stained with PE streptavidin. Quadrants were set so that greater than 98% of the cells were in the lower left quadrant in background stained samples (no fluorescein). PE quadrants were raised in C to delineate the TcR+ population. AKR/J and BALB/c cells were assayed in separate experiments. FI-HA + MEL-14
Background
Three-color flow cytometry of BALB/c T spleen allowed gating on Thy-l+ cells, detected using coumarin-conjugatcd 30H12 antibody, while also staining with Fl-HA and biotin-labeled MEL-14. The small population of Mel14-negative T cells was especially bright for FI-HA staining (Fig. 3). Table 1. CD44-dependent adhesion of splenicT cell to H A and to endothelial cells”)
Balb/c
AKWJ CD44
100
Antibody added
2721
CD44 as an hyaluronic acid receptor on T cells
Eur. J. Immunol. 1992. 22: 2719-2723
FI-fluorescence
Figure 3. Fl-HA binding on MEL-14-positive and -negative BALBk splenic T cclls. Splenic T cells from BALBk mice were stained in (B) with coumarin-conjugated Thy-1 specific antibody 30-H12, biotin-conjugated MEL-14 antibody [13], specific for L-selectin, plus PE-streptavidin, and F1-HA in the presence of IRAWB 14 mAb. “Background” cells (A) were stained with coumarin-30-Hl2, PE-streptavidin, and FI-HA in the presence of IRAWB 14 and excess unlabeled HA. Shown are profiles after gating on Thy-l+ (coumarin-positive) cells.
4 Discussion HA-binding function of CD44 can be demonstrated in some CD44-expressing cell lines and in cells transfected with the hematopoietic form of CD44 [l,3-51. However, most normal hematopoietic cells of the mouse that express
2722
Eur. J. Immunol. 1992. 22: 2719-2723
J. Lesley and R. Hyman
CD44 do not constitutively bind HA.This observation, and the ability to induce H A binding by CD44 under specific conditions (Fee below), has suggested that normal hematopoietic cells regulate the HA-receptor function of CD44 [51. As we show here, normal resting murine Tcells can be “activated” to bind H A upon treatment with certain CD44-specific antibodies, while other hematopoietic cell types are not activated (Fig. 1). We use “activation” to indicate the rapid (nearly instantaneous) conversion of pre-existing CD44 molecules from a non-HA-binding state to a form able to bind HA. B cells,which were not activated by IRAWB 14 mAb, can be induced to bind H A by culture for several days in IL-5 [141or by chronic zn vzvo stimulation through a GVH reaction [6], both of which also result in increased CD44 expression and induction of immunoglobulin secretion. These observations suggest that: (a) both B and T cell? are capable of binding H A through CD44, and (b) the CD44 molecules on resting B and T cells are in different states with respect to HA-binding function. Evidence from both normal murine lymphocytes and lymphoid cell lines suggests that there are three possible states of H A receptor function for CD44: (a) non-activatable, corresponding to resting B cells, some pre-B lymphomas, and bone marrow cells, (b) activatable (rapidly convertible to HA-binding function by IRAWB 14 mAb) represented by resting CD44+ T cells and some T lymphomas [S], and (c) constitutively active, or able to bind H A without activation [3]. Maturatioddifferentiation steps over a period of hours or days, such as exposure to IL-5 for B cell? or other unknown stimuli for other hematopoietic ccll types, may be required to convert non-activatable cells to cells which can be activated to bind HA or which bind H A constitutively. The activatable state could permit rapid and reversible regulation of CD44 HA-receptor function under appropriate circumstance?. The constitutive HAbinding state observed in some T lymphomas and plasmacytomas [ 1, 31, as well as B cells induced by IL-5 and GVH [6, 141, may be the result of transformation of an activated cell or chronic activation. respectively. These states of activation are similar to those described for LFA-1 on lymphocyter by Figdor et al. [15]. The physiological mediators that induce differentiation of non-activatable CD44+ normal hematopoietic cells to a state capable of binding H A or that induce rapid activation are unknown, with the exception of IL-5 stimulation of B cell differentiation. One would predict that activation should be rapid and reversible and, therefore, possibly mediated by signal transducing systems involving phosphorylationfdephosphorylation by protein kinases and phosphatascs, Ca2+ tlux, and/or the inositol phospholipid pathway.Yet, we wcre unable to activate H A binding in T cells with phorbol ester, calcium ionophore, or signaling through CD3 cross-linking. Antibody activation bypasses these pathways in the case of LFA-1 [15] and CD44 (Lesley et al., submitted for publication).
CD44 is elevated in murine memoryT cells and uponT cell activation both in vitro and in vivo [16]. Recent studies indicate that CD8+ cells with a protective effect against malaria infection [17] or which infiltrate a graft site [lS] exprcss high levcls of CD44 and other adhesion receptors. CD44 is also elevated on human memory T cells [19] and
upon in vitro stimulation of human T cells with phorbol ester [20]. Since we show here that the level of CD44 expression correlates closely with the ability to bind H A after IRAWB 14-induced activation of CD44 (Fig. 2), memory and activated T cells are likely to be among those capable of binding HA. The MEL-14 (L-selectin)-negative T cells of the spleen, which include the recently activated and memory T cells [21, 221, show especially high F1-HA binding activity after IRAWB 14 antibody activation (Fig. 3). Normal hematopoietic cells reflect several functional states of CD44 with respect to H A receptor function, examples of which are also found in lymphoid cell lines. Transitions in activation state similar to those we describe for H A binding by CD44 are observed with other cell adhesion molecules including the integrins and selectins [15,23-28]. Understanding the regulation of the adhesion functions of these molecules is crucial to understanding their roles in cell-cell interactions, migration and activation. Received May 14, 1992, in final revised form July 13, 1992.
5 References 1 Miyake, K., Underhill, C. B., Lesley, J. and Kincade, l? W., J. Exp. Med. 1990. 172: 69. 2 Aruffo, A. L., Stamenkovic, L., Melnick. M.; Underhill, C. B. and Seed, B., Cell 1990. 61: 1303. 3 Lesley, J., Schulte, R. and Hyman, R., Exp. Cell. Res. 1990. 187: 224. 4 Stamenkovic, I., Arfurro, A., Amiot, M. and Seed, B., EMBO J. 1991. 10: 343. 5 Lesley, J., He, Q., Miyake, K., Hamann, A., Hyman, R. and Kincade, P. W., J. Exp. Med. 1992. 175: 257. 6 Murakami, S., Miyake, K., Abe, R., Kincade, P.W. andHodes, R. J., J. Immunol. 1991. 146: 1422. 7 Hyman, R., Lesley, J. and Schulte, R.. Immunogenetics 1991. 33: 329. 8 Lesley, J., Hyman, R. and Schulte, R.. Cell. Irnrnunol. 1985.91: 397. 9 Harder, R., Uhlig, H., Kasha, A , , Schutt, B., Duijvestijn, A., Butcher, E. C.,Thiele, H.-G. and Hamann, A.. Exp. Cell Res. 1991. 197: 259. 10 Lesley, J., Schulte, R., Trotter, J. and Hyman, R., Cell. Irnmunol. 1988. 112: 40. 11 Lesley, J., Trotter, J., Schulte, R. and Hyman, R., Cell. Irnrnunol. 1990. 128: 63. 12 Lynch, F. and Ceredig, R., Eur. J. Immunol. 1989. 19: 223. 13 Gallatin,W. M.,Weissman, I. L. and Butcher, E . C., Nature 1983. 304: 30. 14 Murakami, S., Miyake, K., June, C. H., Kincade, l? W. and Hodes, R. J., J. Imrnunol. 1990. 145: 3618. 15 Figdor, C. G.,Van Kooyk,Y. and Keizer, G. D., lmrnunol. Today 1990. 11: 277. 16 Budd, R. C., Cerottini, J.-C., Horvath, C., Bron, C., Pedrazzini,T., Howe, R. C. and MacDonald, H . R., J. Irnrnunol. 1987. 138: 3120. 17 Rodrigues, M.. Nussenzweig, R. S, Romero, P. and Zavala, F., J. Exp. Med. 1992. 175: 895. 18 Mobley, J. L. and Dailey. M. O., J. Immunol. 1992.148: 2348. 19 Sanders, M. E., Makgoba, M.W., Sharrow, S. O., Stephany, D., Springer,T. A.,Young, H. A. and Shaw, S., J. Immunol. 1988. 140: 1401. 20 Oppenheimer-Marks, N., Davis, L. S. and Lipsky, l? E., J. Irnmunol. 1990. 14.5: 140.
Eur. J. Immunol. 1992. 22: 2719-2723
21 Bradley, L. M., Duncan, D. D.,Tonkonogy. S. and Swain, S. L., J. Exp. Med. 1991. 174: 547. 22 Bradley, L. M.. Atkins, G. G. and Swain, S. L., J. Immunol. 1992. 148: 324. 23 Dustin, M. L. and Springer,T. A , , Annu. Rev. Immunol. 1991. 9: 27. 24 0’Toole.T. E., Loftus, J. C., Du, X., Glass, A . A., Ruggeri, Z . M. Shattil, S. J., Plow, E. F. and Ginsberg, M. H., Cell Regul. 1990. I : SS.1.
CD44 as an hyaluronic acid receptor on T cells
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25 Altieri, D. C., Bader. R., Mannucci, F! M. and Edgington,T. S., J. Cell Biol. 1988. 107: 1893. 26 Graham, I. and Brown, E. J., . I . Immunol. 1991. 146: 685. 27 Neugebauer, K. M. and Reichardt, L. F., Nature 1991.350: 68. 28 Spertini, O., Kansas, G. S., Munro, J. M., Griffin, J. D. and Tedder,T. F., Nature 1991. 349: 691.
Eur. J. Immunol. 1992. 22: 2719-2723
Jayne Lesley and Robert Hyman Department of Cancer Biology, The Salk Institute, San Diego
2719
CD44 can be activated to function as an hyaluronic acid receptor in normal murine T cells* The hyaluronic acid (HA)-binding function of CD44 expressed on the cell surface of normal hematopoietic cells has been studied by assaying binding of fluoresceinated hyaluronic acid (FI-HA) and adhesion to immobilized HA. As has been observed previously, normal hematopoietic cells from bone marrow and spleen do not constitutively bind HA. A CD44-specific monoclonal antibody, IRAWB 14, which has been shown to rapidly induce H A binding in some CD44+ cell lines,was used to activate the HA-binding function of CD44 in these normal cells. Only splenic T cells were activated by the IRAWB 14 antibody to bind F1-HA. Upon activation, FI-HA binding correlated with the level of CD44 expression. Activation of H A binding allowed splenicT cells to adhere to H A immobilized on plastic and to an endothelial cell line in an HA-dependent manner. BALBlc and AKR/J splenicT cells differ in their level of CD44 expression, and this correlated with differences in their ability to bind H A upon antibody activation. The minor subpopulation of MEL-14- T cells were among the brightest F1-HA-staining cells. We propose, on the basis of these and other results, that there are three states of CD44 function with respect to H A binding: (a) a non-activatable, resting state, which cannot be rapidly activated to bind H A , as seen in most hematopoietic cells; (b) an activatable state, which can be rapidly converted to HA-binding function, in this case by the IRAWB 14 antibody, illustrated by Tcells as shown here; and (c) a constitutively active state, which can bind H A without antibody activation, seen in some cell lines.
1 Introduction In vitvo, CD44 has been shown to be a receptor for hyaluronic acid (HA), a high-molecular weight, repeating disaccharide, that is present in extracellular matrix and body fluids [ 1-51. However, most normal hematopoietic cells of the mouse which express CD44 do not constitutively bind H A in vitro [3, 5 , 61. We have described a CD44specific mAb, IRAWB 14, which rapidly “activates” HAbinding function in some CD44+, HA-receptor- cell lines [5].We use the tcrm “activation” to indicate the conversion of pre-existing CD44 molecules from a non-HA-binding state to a form able to bind HA.This is distinguished from the effects of phorbol esters on some T lymphomas, which occur over several hours and are accompanied by increased CD44 expression [3, 71. Here, we show that the CD44specific mAb, IRAWB 14, also activates HA-receptor function in normal murine T cells that express CD44. Differences in the level of CD44 expressed on T cells of BALB/c mice and AKR/J mice correlate with the HA-
binding capacity of these cells. Splenic B cells and bone marrow cells are not activated t o bind H A by the IRAWB 14 antibody.
2 Materials and methods 2.1 Cell preparation
Single cell suspensions of spleen and bone marrow were prepared from AKR/J and BALB/c J mice (Jackson Laboratories, Bar Harbor, ME).T cells were enriched from spleen by panning on petri dishes coated with antibody t o immunoglobulin. After panning 650/0-850/0 of the recovered nonadherent cells were shown to beT cells on the basis of staining with fluoresceinated antibodies against Thy-1 alleles. B cells were enriched from spleen by two rounds of cytotoxic depletion with cytotoxic Thy-1-specific antibodies and rabbit complement, and recovery of live cells by centrifugation over Lymphoprep (Nycomed AS, Oslo, Norway) [S]. These populations contained no cells staining with fluoresceinated anti-Thy-1 antibodies. The murine LN endothelial cell line, TME-3H3 [5, 91, was harvested with trypsin and replated at 1 x lo4 -2 x 104/wellon 24-well [I 106251 plates 1 or 2 days before use in adhcsion assays.
* This work was wpportcd by National Cancer Institute Grant CA-13287 and Natmnal Institute of Allergy and Infectious Diseaxs Grant AT-31613 to R. Hyman, by National Cancer Institute Core Grant CA-14195 to the Salk Institute and by thc Hansen Foundation. Correspondence: Jayne Lesley, Department of Cancer Biology, The Salk Institutc, PO. Box 85800, San Diego, CA 92186-5800, USA Abbreviations: HA: Hyaluronic acid FI-HA: Fluorescein-conjugated hyaluronic acid
0 VCH Vcrlagsgesellschaft mbH, D-6940 Weinheim, 1992
2.2 Flow cytometry
Cell surface antigen expression was determined by flow cytometry using a FACScan (for one- and two-color analysis) or FACStar+ (for three-color analysis) (Becton Dickinson, Mountain View, CA). Antibodies and cell staining are as described in [lo] and [ 111, and in the Figure legends. Coumarin-labeled 30H12 was used for three-color analysis [ll]of BALB/c spleenT cells, to allow gating o n T 0014-2980/92/1010-2719$3.50+ .25/0
2720
J. Leslcy and R. Hyman
cells, and staining with FI-HA and biotinylated antibody plus PE-streptavidin. Fluorescein-conjugated hyaluronic acid (Fl-HA) was prepared and used as previously described [3]. Activation of Fl-HA binding was achieved by simultaneous addition of S pg/ml of purified IRAWB 14 mAb [5]. Specificity of Fl-HA binding was confirmed by blocking with an excess of unlabeled H A (100 pg/ml final concentration).
2.3 Adhesion assays Adhesion assays using T-enriched spleen cells were performed as previously described [5] using 24-well plastic plates coated with HA or bearing monolayers of the TME-3H3 lymph node endothelial line [9]. mAb (as a 1: 20 dilution of hybridoma culture supernatant) were added to Tr-labeled, T-enriched spleen cells just prior to their addition to washed, substrate-coated plates. Percent adhesion was calculated as: cpm adhering + (input cpm spontaneously released cpm). HA dependence of adhesion to TME-3H3 cells was determined by hyaluronidase treatment of TME-3H3 monolayers [ S ] .
Eur. J. Immunol. 1992. 22: 2719-2723 The mean fluorescence of the induced population was only about two-fold greater than the mean of the same population in the presence of unlabeled HA. In an effort to identify other agents capable of rapidly inducing HA-binding activity in T cells, in a manner that would mimic the effect of TRAWB 14 antibody, AKR/J and BALB/c splenic T cells were incubated at 37 "C for 20 min with the following reagents: the phorbol ester PMA, the Ca2+ ionophore ionomycin, PMA ionomycin, Con A, PMA-induced EL4 cell supernatant as a source of IL-2, CD3-specific antibody cross-linked with antibody to hamster immunoglobulin, IL-3, IL-4 and IL-7. FI-HA was then added for 15 min at 4 "C and the cells were subsequently washed and examined by flow cytometry. None of these treatments affected Fl-HA binding by the treated T cells. Culture of splenic T cells overnight in PMA also did not induce binding of F1-HA ([3] and data not shown).
+
FL-anti-CD44
FL-HA alone
FL-HA+IRAWB14
3 Results 3.1 Normal murine T cells, but not other CD44+ hematopoietic cells, can be induced to bind Fl-HA Cell populations prepared from spleen and bone marrow of AKR/J and BALB/c mice were stained with fluoresceinconjugated TM7 mAb (to detect CD44 expression) and with Fl-HA and analyzed by flow cytometry (Fig. 1). These mouse strains represent two extremes in the level of CD44 expression on thymocytes and T cells [lo, 121. Column 1in Fig. 1 illustrates this difference in expression of CD44 on peripheral T cells.While AKR/J Tspleen contains a CD44low or -negative population, a CD44-intermediate population and a CD44-high population, BALBkTspleen cells all express relatively high levels of CD44. B cells in both BALBk and AKR/J mice express uniformly high levels of CD44, but AKR/J bone marrow contains a CD44-negative population not present in BALB/c bone marrow. When the binding of Fl-HA by these populations is examined (Fig. 1, center column), using blocking by unlabeled H A (dotted curve) as the criterion for specificity, no specific HA binding by any of the populations can be demonstrated. However, in the presence of IRAWB 14 antibody, a distinct population of F1-HA binding T cells is seen (Fig. 1, last column, first and second panels). This binding is competed by unlabeled H A (dotted insert). In spleen from AKR/J mice, the F1-HA-positive population only contains about one third of the T cells, corresponding to the brightest CD44-staining population (see below), while in BALB/c spleen, most T cells are positive at some level. Splenic B cell-enriched populations and total bone marrow in both mouse stains showed a slight positive shift in Fl-HA staining in the presence of IRAWB 14,which was competed by unlabeled H A (Fig. 1, panels 3-6 in the last column).
I
B Cells
Balb/c Splenic B Cells
!/;,..A1 I
1
Relative Fluorescence Intensity
Figure 1. Expression and HA binding function of normal hematopoietic cells: T-enriched spleen cells (rows 1 and 2), B-enriched spleen cells (rows 3 and 4), and total bone marrow (rows 5 and 6). In the first column, CD44 expression was determined using FITC-conjugated CD44-specific antibody IM7. Dotted insert is the background fluorescence of unstained cells. In the second column, cells were stained with FI-HA.The dotted insert is F1-HA staining in the presence of excess unlabeled HA. In the third column, cells were stained with FI-HA in the presence of 5 Kgglml of purified IRAWB 14 antibody. The dotted insert is FI-HA staining in the presence of IRAWB 14 and excess unlabeled HA.
3.2 Hyaluronic acid-dependent adhesion can be activated in T cells AKR/J T-enriched spleen cells were used to study induction of cell adhesion to hyaluronic acid. Cells were treated with CD44-specific mAb KM201, which inhibits CD44-dependent binding of H A [l], or with CD44-specific mAb IRAWB 14, which activates CD44-dependent binding of F1-HA, and the treated cells were assayed for adhesion to plastic wells coated with H A or to the endothelial cell line TME-3H3 (Table 1). There was very little adhesion to HA-coated plates without induction. IRAWB 14 mAb activated significant adhesion to HA-coated culture wells and caused increased adhesion to endothelial cells. As observed previously with cell lines [5], a portion of the binding of untreated cells to endothelial cells was not CD44 dependent, since it could not be inhibited by KM201 mAb. Hyaluronidase treatment of TME-3H3 cells abolished the increase in T cell adhesion that was induced by IRAWB 14 an ti body (not shown) .
3.3 F’l-HA binding correlates directly with CD44 expression In two-color flow cytometric analysis of splenic T cells, F1-HA binding cells (induced in the presence of IRAWB 14 mAb) were also stained with biotin-labeled antibodies against other cell surface antigens plus PE-labeled streptavidin. In both AKR/J spleen and BALB/c spleen, the intensity of F1-HA staining was directly proportional to the level of CD44 expression (Fig. 2 A, B). T cells were specifically identified by the expression of TcR (Fig. 2 C, D), CD4 or CD8. A proportion of CD4+ and CD8+ cells were positive for FI-HA in both strains. However, in BALB/c spleen, the majority of CD4+ cells were positive for Fl-HA staining, while in AKR/J spleen most of the CD4+ cells did not bind F1-HA or bound very weakly (data not shown). The difference in Fl-HA staining among CD4+ cells accounts for much of the difference in F1-HA binding in BALB/c splenic T cells versus AKR/J splenic T cells.
103
8
g
102
0
?
s
10’
W
a 100
TCR
None
KM201b) IRAWB 14
YOadhesion to HA plates
% adhesion to 3H3 cells
6.9% k 0.1 2.8% k 0.3 34.0% k 7.8
25.4% k 4.0 14.5% k 3.1 44.5% k 2.2
a) Mean and standard error of four replicate samples. b) KM201 i s a CD44-specific mAb that has been shown to inhibit CD44 binding to H A [I]. Binding in the presence of KM201 antibody (not inhibited by KM201) is considered to be nonCD44 dependent (ie., nonspecific, or due to specific factors other than CD44).
D
C
103
I
io1
10‘
103
. . . . .J...? : 101
100
,....,.....,......
102
103
FI-HA fluorescence
Figure 2. Relationship of IRAWB 14-induced F1-HA binding to expression of CD44 and Tcell receptor. F1-HA binding was induced on splenic T cells by treatment with mAb IRAWB 14. CD44 was detected with biotinylated-IM7 antibody (A and B) and TcR was detected with biotinylated -57.597 (specific for afi TcR; C and D). Biotin-antibodies were stained with PE streptavidin. Quadrants were set so that greater than 98% of the cells were in the lower left quadrant in background stained samples (no fluorescein). PE quadrants were raised in C to delineate the TcR+ population. AKR/J and BALB/c cells were assayed in separate experiments. FI-HA + MEL-14
Background
Three-color flow cytometry of BALB/c T spleen allowed gating on Thy-l+ cells, detected using coumarin-conjugatcd 30H12 antibody, while also staining with Fl-HA and biotin-labeled MEL-14. The small population of Mel14-negative T cells was especially bright for FI-HA staining (Fig. 3). Table 1. CD44-dependent adhesion of splenicT cell to H A and to endothelial cells”)
Balb/c
AKWJ CD44
100
Antibody added
2721
CD44 as an hyaluronic acid receptor on T cells
Eur. J. Immunol. 1992. 22: 2719-2723
FI-fluorescence
Figure 3. Fl-HA binding on MEL-14-positive and -negative BALBk splenic T cclls. Splenic T cells from BALBk mice were stained in (B) with coumarin-conjugated Thy-1 specific antibody 30-H12, biotin-conjugated MEL-14 antibody [13], specific for L-selectin, plus PE-streptavidin, and F1-HA in the presence of IRAWB 14 mAb. “Background” cells (A) were stained with coumarin-30-Hl2, PE-streptavidin, and FI-HA in the presence of IRAWB 14 and excess unlabeled HA. Shown are profiles after gating on Thy-l+ (coumarin-positive) cells.
4 Discussion HA-binding function of CD44 can be demonstrated in some CD44-expressing cell lines and in cells transfected with the hematopoietic form of CD44 [l,3-51. However, most normal hematopoietic cells of the mouse that express
2722
Eur. J. Immunol. 1992. 22: 2719-2723
J. Lesley and R. Hyman
CD44 do not constitutively bind HA.This observation, and the ability to induce H A binding by CD44 under specific conditions (Fee below), has suggested that normal hematopoietic cells regulate the HA-receptor function of CD44 [51. As we show here, normal resting murine Tcells can be “activated” to bind H A upon treatment with certain CD44-specific antibodies, while other hematopoietic cell types are not activated (Fig. 1). We use “activation” to indicate the rapid (nearly instantaneous) conversion of pre-existing CD44 molecules from a non-HA-binding state to a form able to bind HA. B cells,which were not activated by IRAWB 14 mAb, can be induced to bind H A by culture for several days in IL-5 [141or by chronic zn vzvo stimulation through a GVH reaction [6], both of which also result in increased CD44 expression and induction of immunoglobulin secretion. These observations suggest that: (a) both B and T cell? are capable of binding H A through CD44, and (b) the CD44 molecules on resting B and T cells are in different states with respect to HA-binding function. Evidence from both normal murine lymphocytes and lymphoid cell lines suggests that there are three possible states of H A receptor function for CD44: (a) non-activatable, corresponding to resting B cells, some pre-B lymphomas, and bone marrow cells, (b) activatable (rapidly convertible to HA-binding function by IRAWB 14 mAb) represented by resting CD44+ T cells and some T lymphomas [S], and (c) constitutively active, or able to bind H A without activation [3]. Maturatioddifferentiation steps over a period of hours or days, such as exposure to IL-5 for B cell? or other unknown stimuli for other hematopoietic ccll types, may be required to convert non-activatable cells to cells which can be activated to bind HA or which bind H A constitutively. The activatable state could permit rapid and reversible regulation of CD44 HA-receptor function under appropriate circumstance?. The constitutive HAbinding state observed in some T lymphomas and plasmacytomas [ 1, 31, as well as B cells induced by IL-5 and GVH [6, 141, may be the result of transformation of an activated cell or chronic activation. respectively. These states of activation are similar to those described for LFA-1 on lymphocyter by Figdor et al. [15]. The physiological mediators that induce differentiation of non-activatable CD44+ normal hematopoietic cells to a state capable of binding H A or that induce rapid activation are unknown, with the exception of IL-5 stimulation of B cell differentiation. One would predict that activation should be rapid and reversible and, therefore, possibly mediated by signal transducing systems involving phosphorylationfdephosphorylation by protein kinases and phosphatascs, Ca2+ tlux, and/or the inositol phospholipid pathway.Yet, we wcre unable to activate H A binding in T cells with phorbol ester, calcium ionophore, or signaling through CD3 cross-linking. Antibody activation bypasses these pathways in the case of LFA-1 [15] and CD44 (Lesley et al., submitted for publication).
CD44 is elevated in murine memoryT cells and uponT cell activation both in vitro and in vivo [16]. Recent studies indicate that CD8+ cells with a protective effect against malaria infection [17] or which infiltrate a graft site [lS] exprcss high levcls of CD44 and other adhesion receptors. CD44 is also elevated on human memory T cells [19] and
upon in vitro stimulation of human T cells with phorbol ester [20]. Since we show here that the level of CD44 expression correlates closely with the ability to bind H A after IRAWB 14-induced activation of CD44 (Fig. 2), memory and activated T cells are likely to be among those capable of binding HA. The MEL-14 (L-selectin)-negative T cells of the spleen, which include the recently activated and memory T cells [21, 221, show especially high F1-HA binding activity after IRAWB 14 antibody activation (Fig. 3). Normal hematopoietic cells reflect several functional states of CD44 with respect to H A receptor function, examples of which are also found in lymphoid cell lines. Transitions in activation state similar to those we describe for H A binding by CD44 are observed with other cell adhesion molecules including the integrins and selectins [15,23-28]. Understanding the regulation of the adhesion functions of these molecules is crucial to understanding their roles in cell-cell interactions, migration and activation. Received May 14, 1992, in final revised form July 13, 1992.
5 References 1 Miyake, K., Underhill, C. B., Lesley, J. and Kincade, l? W., J. Exp. Med. 1990. 172: 69. 2 Aruffo, A. L., Stamenkovic, L., Melnick. M.; Underhill, C. B. and Seed, B., Cell 1990. 61: 1303. 3 Lesley, J., Schulte, R. and Hyman, R., Exp. Cell. Res. 1990. 187: 224. 4 Stamenkovic, I., Arfurro, A., Amiot, M. and Seed, B., EMBO J. 1991. 10: 343. 5 Lesley, J., He, Q., Miyake, K., Hamann, A., Hyman, R. and Kincade, P. W., J. Exp. Med. 1992. 175: 257. 6 Murakami, S., Miyake, K., Abe, R., Kincade, P.W. andHodes, R. J., J. Immunol. 1991. 146: 1422. 7 Hyman, R., Lesley, J. and Schulte, R.. Immunogenetics 1991. 33: 329. 8 Lesley, J., Hyman, R. and Schulte, R.. Cell. Irnrnunol. 1985.91: 397. 9 Harder, R., Uhlig, H., Kasha, A , , Schutt, B., Duijvestijn, A., Butcher, E. C.,Thiele, H.-G. and Hamann, A.. Exp. Cell Res. 1991. 197: 259. 10 Lesley, J., Schulte, R., Trotter, J. and Hyman, R., Cell. Irnmunol. 1988. 112: 40. 11 Lesley, J., Trotter, J., Schulte, R. and Hyman, R., Cell. Irnrnunol. 1990. 128: 63. 12 Lynch, F. and Ceredig, R., Eur. J. Immunol. 1989. 19: 223. 13 Gallatin,W. M.,Weissman, I. L. and Butcher, E . C., Nature 1983. 304: 30. 14 Murakami, S., Miyake, K., June, C. H., Kincade, l? W. and Hodes, R. J., J. Imrnunol. 1990. 145: 3618. 15 Figdor, C. G.,Van Kooyk,Y. and Keizer, G. D., lmrnunol. Today 1990. 11: 277. 16 Budd, R. C., Cerottini, J.-C., Horvath, C., Bron, C., Pedrazzini,T., Howe, R. C. and MacDonald, H . R., J. Irnrnunol. 1987. 138: 3120. 17 Rodrigues, M.. Nussenzweig, R. S, Romero, P. and Zavala, F., J. Exp. Med. 1992. 175: 895. 18 Mobley, J. L. and Dailey. M. O., J. Immunol. 1992.148: 2348. 19 Sanders, M. E., Makgoba, M.W., Sharrow, S. O., Stephany, D., Springer,T. A.,Young, H. A. and Shaw, S., J. Immunol. 1988. 140: 1401. 20 Oppenheimer-Marks, N., Davis, L. S. and Lipsky, l? E., J. Irnmunol. 1990. 14.5: 140.
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21 Bradley, L. M., Duncan, D. D.,Tonkonogy. S. and Swain, S. L., J. Exp. Med. 1991. 174: 547. 22 Bradley, L. M.. Atkins, G. G. and Swain, S. L., J. Immunol. 1992. 148: 324. 23 Dustin, M. L. and Springer,T. A , , Annu. Rev. Immunol. 1991. 9: 27. 24 0’Toole.T. E., Loftus, J. C., Du, X., Glass, A . A., Ruggeri, Z . M. Shattil, S. J., Plow, E. F. and Ginsberg, M. H., Cell Regul. 1990. I : SS.1.
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25 Altieri, D. C., Bader. R., Mannucci, F! M. and Edgington,T. S., J. Cell Biol. 1988. 107: 1893. 26 Graham, I. and Brown, E. J., . I . Immunol. 1991. 146: 685. 27 Neugebauer, K. M. and Reichardt, L. F., Nature 1991.350: 68. 28 Spertini, O., Kansas, G. S., Munro, J. M., Griffin, J. D. and Tedder,T. F., Nature 1991. 349: 691.
