Regulation of T Cell Homeostasis by Heparan Sulfate-Bound IL-2¹

For many years, the extracellular matrix was thought to be an inert scaffold, with cellular functions taking place independent of the surrounding stroma. Today it is well established that components of the extracellular matrix play an integral role in shaping cell behavior. Cell-matrix interactions may occur via the retention by the extracellular matrix of soluble mediators released by cells. This sequestration alters local concentrations of these mediators and thus modulates cell functions.

Several mediators, particularly chemokines, are localized within the extracellular matrix through binding to heparan sulfate, a polysaccharide consisting of repeating disaccharides glucosamine and hexuronic acid, covalently linked to a protein core (1). The saccharides are extensively modified by N-sulfation, O-sulfation, and epimerization of glucuronic acid, resulting in significant heterogeneity within the saccharide chains (2, 3). These chains likely confer biological activity upon the proteoglycan, and constitute the binding site for the various mediators.

Many of the substances that bind heparan sulfate are produced by cells of the immune system. Chemokines such as IL-8, RANTES, macrophage-inflammatory protein-1β (MIP-1β),³ and hepatocyte growth factor attract passing immune cells and alter their level of activation (4, 5, 6). Cytokines such as GM-CSF (7), IL-3 (8), IL-4 (9), IFN-γ (10), IL-7 (11), and IL-2 (12) bind heparan sulfate as well. Although interactions between heparan sulfate and mediators such as chemokines/cytokines likely have significant biological consequences, many of the studies reporting these interactions have been performed in vitro, and thus the functional portent of these associations in vivo are incompletely understood.

Whether these heparan sulfate-bound mediators are utilized by their target cells in a bound vs soluble form is not yet established for each mediator. Metalloproteinases (13), heparanases (14), or substances such as NO (15), may cleave heparan sulfate glycosaminoglycan chains, thus releasing the mediators in soluble form. These enzymes may be produced during the course of an immune response by activated T cells, neutrophils, and macrophages (14, 16, 17). In some cases (such as with fibroblast growth factor, IFN-γ, and IL-8), the growth factor involved remains bound to heparan sulfate, and through this association the bioactivity of that growth factor is modified (4, 10, 18).

IL-2, also reported to bind heparan sulfate, regulates many aspects of T cell function. IL-2 promotes proliferation of naive T cells that encounter Ag and memory T cells that reencounter Ag following an interval return to a resting state (19). On the other hand, IL-2 promotes apoptosis of activated T cells reexposed to Ag by sensitizing these cells to fas-mediated cell death (20). Finally, IL-2 prevents “passive cell death” via apoptosis in naive or activated T cells, which will otherwise die if cytokine support is withdrawn (21). Other cytokines using the common γ-chain of the IL-2 receptor may substitute for IL-2 in enhancing T cell survival, but to date no other cytokines have been shown to efficiently replace IL-2 in its effects on activation-induced cell death (22). (Other cytokines have been reported to prime T cells for fas-mediated cell death, but much higher concentrations are required (23, 24)). The critical role of IL-2 in T cell homeostasis is demonstrated by the phenotype of IL-2-deficient mice, which develop splenomegaly, lymphocytosis, and autoimmune-mediated disorders including hemolytic anemia and colitis (25).

Although IL-2 binds heparan sulfate in vitro, whether this interaction actually occurs in vivo and how it might impact immune responses is unknown. We therefore asked whether IL-2 in fact binds heparan sulfate in vivo, and what effects such binding might have on T cell responses. Here we show that heparan sulfate tethers IL-2 in lymphoid organs and that heparan sulfate-associated IL-2 promotes T cell proliferation and apoptosis in vivo.

DO11.10 TCR transgenic, IL-2^+/+ BALB/c mice, DO11.10 TCR transgenic, IL-2^−/− BALB/c mice (both generously provided by Dr. Marc Jenkins (University of Minnesota, Minneapolis, MN), and C57BL/6 IL-2^−/− mice were bred in our facility under specific pathogen-free conditions. BALB/c^nu/nu mice were purchased from B & K (Kent, WA).

KJ1-26 (mouse IgG), a mAb against the ova-specific T cell receptor was generously provided by Dr. Marc Jenkins. R14, a rabbit polyclonal anti-perlecan Ab, was generously provided by Dr. Alan Snow and Dr. Geraldo Castillo (Proteotech, Seattle, WA). Biotinylated TCR anti-Vβ3, biotinylated TCR anti-Vβ8, cychrome-labeled anti-CD4, rat anti-mouse IL-2 (S4B6), and appropriate secondary Abs (listed below) were obtained from PharMingen (San Diego, CA). Anti-heparan sulfate Abs (10E4) and anti-chondroitin sulfate Abs (clone MO-225) were purchased from Seikagaku (Tokyo, Japan).

Tissues were snap-frozen in precooled isopentane, cut into 4-μ sections, and fixed briefly in acetone. Cut sections were then stained with rat anti-mouse IL-2 diluted in PBS with 0.1% saponin, followed by fluorescein-labeled goat anti-rat and swine anti-goat Abs. No staining was detectable with an isotype control as the primary Ab, with secondary Abs alone, or with rat anti-mouse IL-2 Abs preincubated with murine IL-2 prior to use.

Additional sections were stained simultaneously for IL-2 and perlecan. Perlecan was detected using a rabbit polyclonal anti-perlecan Ab followed by a TRITC-labeled goat anti-rabbit secondary Ab. Sections stained with anti-rabbit Abs alone were negative. Sections stained by reversing the secondary Abs were negative, and sections stained individually for either perlecan or IL-2 were identical to the double-labeled sections.

Tissues were analyzed for apoptosis via the TUNEL method (26) by incubation with terminal deoxynucleotidyltransferase and FITC-labeled dUTP (Oncor, Gaithersburg, MD). Tissue sections digested with enzymes were left unfixed then treated with 8 U/ml heparitinase I or chondroitinase (Sigma, St. Louis, MO) in 50 mM NaCl and 1 μM CaCl (pH 7.0) for 2 h at 30°C. The sections were subsequently washed, fixed in acetone, then stained for IL-2.

Cells isolated from lymph nodes and spleen were stained with biotinylated anti-vβ3 or anti-vβ8 followed by PE-labeled streptavidin, and rat anti-mouse CD4 directly labeled with CyChrome. In the adoptive transfer experiments, cells were stained with KJ1-26 followed by FITC-labeled streptavidin and CD4-PE. Stained cells were then analyzed for the frequency of double-positive cells using a FACScan (Becton Dickinson, San Jose, CA).

Heparan sulfate glycosaminoglycan was extracted from human spleen (27, 28) and captured onto microtiter plates preincubated with anti-heparan sulfate Abs (2 μg/ml), yielding a final concentration of immobilized heparan sulfate glycosaminoglycan of approximately 10 μg/well. Control wells were treated with an anti-chondroitin sulfate Ab (clone MO-225, Seikagaku; 2 μg/well) followed by incubation with human chondroitin sulfate glycosaminoglycans (20 μg/well; also extracted from spleen) yielding a final adsorption of approximately 10 μg of chondroitin sulfate glycosaminoglycan/well. Iodinated human IL-2 (NEN, Boston, MA) was serially diluted in PBS and each dilution (in duplicate) was incubated overnight at 4°C with immobilized heparan- or chondroitin sulfate glycosaminoglycan. Following the incubation, unbound IL-2 was removed by multiple washes, and the bound IL-2 remaining was solubilized with 2 M NaOH and counted in a scintillation counter.

To assess the biological properties of heparan sulfate-bound IL-2, human IL-2 (600 U/ml) or PBS was incubated overnight at 4°C with the immobilized heparan sulfate glycosaminoglycan (or anti-heparan sulfate Ab only) and free IL-2 was removed by washing. The proliferative response of the IL-2-dependent cell line CTLL-2, plated at a concentration of 5000 cells/well, was assessed 24 h later based on incorporation of tritiated thymidine.

C57BL/6, IL-2-deficient mice were given either 125,000 U IL-2 or PBS via tail vein 24 h prior to the administration of Ag. Staphyloccal enterotoxin A (SEA), 10 μg, was then administered i.p. to initiate activation-induced cell death. Six days later, lymph nodes were harvested from the peripheral (axillary, brachial, and cervical lymph nodes) and mesenteric (perihepatic lymph nodes) circulations for FACS analysis. For assessment of IL-2-primed activation-induced cell death in “IL-2 replete” T cells, OVA-specific T cells from BALB/c DO11.10 TCR transgenic mice were cultured with ova 1 mg/ml for 48 h. After harvesting and washing, 2.5 × 10⁶ OVA-specific T cells were injected via tail vein (see Fig. 5,E) or i.p. (Fig. 5 F) into BALB/c nude mice that were infused 24 h previously with 125,000 U IL-2 or PBS. Approximately 2 h later, the infused T cells were restimulated in vivo by the i.p. injection of 2 mg OVA. The frequency of OVA-specific (KJ1-26⁺CD4⁺) T cells in peripheral lymph nodes and spleen was assessed by FACS analysis 6 days later.

For the assessment of proliferative responses induced by heparan sulfate-bound IL-2, BALB/c nude mice were given 1.0 μg IL-2 or PBS i.p. Thirty hours later, the mice were infused with 2.5 × 10⁶ IL-2-deficient, OVA-specific T cells from BALB/c mice. Seventy-two hours after the infusion of cells, the mice were given 2 mg OVA i.p., and the frequency of OVA-specific (KJ1-26⁺CD4⁺) T cells in peripheral lymph nodes and spleen was assessed by flow cytometry 3 days later.

To determine whether IL-2 might associate with elements of the extracellular matrix in vivo, we examined various tissues from mice for evidence of immobilization of IL-2. IL-2 was detected by immunofluorescent microscopy in the perifollicular regions of the spleen, the thymic medulla, along the sinusoids in the liver, and outlining the tubules and within the glomeruli of the kidney (Fig. 1, A to D). IL-2 in lymph nodes, however, was barely detectable or absent (not shown). The specificity of staining for IL-2 was confirmed by the finding that fluorescence was not seen in tissues from mutant mice deficient in IL-2 (Fig. 1,E), and that infusion of IL-2 into IL-2-deficient mice or nude mice (the spleens of which have no detectable perifollicular IL-2) reconstituted the perifollicular pattern of staining seen in the spleens of wild-type animals (Fig. 1 F). IL-4 and IFN-γ, which have also been shown to bind heparan sulfate, were not detected in the extracellular areas of the spleen in naive BALB/c mice.

The tissue distribution described above suggested that IL-2 might be bound to component(s) of the extracellular matrix. Given the prominent staining of the glomeruli and endothelium (areas of increased heparan sulfate concentration) for IL-2, and in light of previous reports demonstrating that fucoidan and heparin bind IL-2 (12, 29, 30), we asked whether IL-2 binds specifically to heparan sulfate. In order to determine whether IL-2 binds heparan sulfate, tissue sections from murine spleen were treated with heparitinase, which specifically cleaves heparan sulfate, prior to staining for IL-2. Digestion of heparan sulfate with heparitinase reduced the binding of IL-2 Abs to tissue sections from spleen (Fig. 1,G), whereas buffer alone had little effect (Fig. 1 H). The specificity of this interaction was demonstrated by digestion with chondroitinase (which cleaves chondroitin sulfate, another acidic glycosaminoglycan), which had little effect (data not shown).

To evaluate directly the potential association of IL-2 with heparan sulfate, we measured the binding of iodinated IL-2 to heparan sulfate glycosaminoglycan extracted from human spleen. Heparan sulfate or chondroitin sulfate glycosaminoglycan was captured onto plastic wells by precoating the wells with anti-heparan sulfate or anti-chondroitin sulfate mAbs (see Materials and Methods). Binding of radiolabeled IL-2 to heparan sulfate was approximately 10-fold higher than binding to chondroitin sulfate (Fig. 2 A), indicating that this interaction is specific to heparan sulfate and is not a nonspecific association based on charge. Similar studies demonstrated that IL-2 also binds heparan sulfate proteoglycan in vitro; this association was inhibited by 90% after preincubation of IL-2 with 100 μg/ml heparin (not shown).

We next asked whether the location of IL-2 in lymphoid organs corresponded to the location of known proteoglycans. Several species of heparan sulfate proteoglycan have been well characterized, including perlecan, the dominant heparan sulfate proteoglycan in basement membranes. To examine the distribution of perlecan in the spleen and its potential correlation with IL-2, splenic sections were double labeled with polyclonal anti-perlecan and anti-IL-2 mAbs. As seen in Fig. 2, B and C, the distribution of perlecan staining significantly overlapped with the pattern of IL-2 staining in the spleen. Both IL-2 and perlecan localized to the red pulp, with a paucity of staining within the follicles. On the other hand, the smooth muscle of intrafollicular arterioles (see upper right hand corner, Fig. 2 B), which are not directly exposed to IL-2-secreting cells, were intensely stained for perlecan but not for IL-2. These data, taken together with the histological studies and in vitro analysis described above, suggest that IL-2 may be tethered to the splenic parenchyma through an interaction with the heparan sulfate glycosaminoglycan chains of perlecan.

To ascertain whether IL-2 is functional when bound to heparan sulfate, we tested the proliferative response of an IL-2-dependent cell line (CTLL-2) to heparan sulfate-bound IL-2. Heparan sulfate glycosaminoglycan was captured onto plastic wells as described above. Following a 24-h incubation of the coated wells with soluble IL-2, excess IL-2 was removed by washing. CTLL-2 cells were then added to the wells and proliferation was analyzed based on incorporation of [³H]thymidine. As Fig. 3 shows, CTLL-2 cells added to wells containing heparan sulfate plus bound IL-2 proliferated, but cells added to wells containing Ab alone or heparan sulfate glycosaminoglycan alone did not. CTLL-2 cells added to wells containing chondroitin sulfate instead of heparan sulfate did not respond (data not shown). Furthermore, these results were not due to a nonspecific association of IL-2 with the anti-heparan sulfate Ab, as CTLL-2 cells did not proliferate in wells sequentially incubated with Ab and IL-2.

We next asked whether intrasplenic levels of IL-2 might be altered by manipulating IL-2 production in vivo. To increase intrasplenic IL-2, mice were administered Con A. Deposition of IL-2 in the spleen was assessed by immunofluorescent microscopy. As seen in Fig. 4, A and B, administration of 500 μg Con A substantially increased the intensity of IL-2 staining in the spleen. In a series of five experiments, the kinetics of IL-2 accumulation varied somewhat, but most commonly the intensity of intrasplenic IL-2 staining appeared greatest at 7 h, declined to a barely detectable level of staining by 24 h, then gradually increased again to a level of intensity consistent with naive spleen over the next 3 to 4 days. In all cases, IL-2 persisted in tissue much longer than IL-2 in blood, which has a serum half-life of 40–80 min (31).

To confirm whether splenic localization of IL-2 requires heparan sulfate, we asked whether administration of heparin might alter binding of IL-2 in the spleen. BALB/c mice were given 160 μg/ml heparin or PBS s.c. in two separate doses 16 h and 1 h prior to the administration of 500 μg Con A. Spleens were harvested 7 h after the administration of Con A and stained for IL-2. Administration of heparin significantly diminished the IL-2 content of the spleen (Fig. 4 C), whereas injection of PBS had no effect. This result likely reflects a competitive displacement of IL-2 by heparin rather than an inhibition of IL-2 production or an influence on homing of T lymphocytes, as baseline levels of intrasplenic IL-2 in naive animals were diminished by heparin as well (not shown). The latter findings confirm that IL-2 interacts with heparan sulfate in vivo, and offer a potential means for manipulating splenic IL-2 stores for immune modulation.

We next asked whether heparan sulfate-bound IL-2, in vivo, might exhibit some of the functions of soluble IL-2. To this end, we developed mice with or without heparan sulfate-bound IL-2, but lacking soluble IL-2, by “reconstituting” the matrices of IL-2-deficient mice. IL-2 (or PBS as a control) was administered systemically 24 h prior to any experimental manipulation of the mice. Twenty-four hours after the injection of 1 μg IL-2, no soluble IL-2 remained in the mice as assessed by several means. The mice had no IL-2 detected in the serum by ELISA (lower limits of detection 0.02 ng/ml) or by bioassay (lower limits of detection 1.75 pg/ml). Nor did the mice have trace levels of circulating IL-2, as [¹²⁵I]IL-2 injected 24 h prior was not detectable in the serum. Despite a lack of soluble IL-2 24-h postinjection, the spleens, assessed by immunofluorescence, contained a significant level of IL-2 (see Fig. 1 F).

We then asked whether heparan sulfate-bound IL-2 might regulate T cell numbers by priming T lymphocytes for activation-induced cell death. We therefore reconstituted the matrices of IL-2-deficient mice with IL-2 and then administered SEA to induce activation-induced cell death. Superantigens such as SEA cause activation-induced death of T cells, which bear the specific β-chain of the TCR recognizing the murine MHC class II—superantigen complex of interest (32). This process is IL-2 dependent as superantigen-stimulated cell death is defective in mice lacking IL-2 Rα or IL-2 (33, 34).

Following reconstitution with IL-2, the mice were inoculated with SEA and frequencies of SEA-specific T cells (Vβ3⁺CD4⁺) or control T cells (Vβ8⁺CD4⁺) were measured 6 days later. As seen in Fig. 5, A and B, the total number of Vβ3⁺ T cells (within the CD4⁺ population) in peripheral lymph nodes and perihepatic lymph nodes of mice reconstituted with IL-2 was approximately threefold lower than mice given PBS. The frequency of Vβ8⁺ T cells was unaffected, indicating that the influence of heparan sulfate-bound IL-2 was specific to Ag-stimulated T cells. These results are consistent with the possibility that heparan sulfate-bound IL-2 is able to reconstitute the ability of IL-2-deficient mice to prime T cells for activation-induced T cell death.

To confirm that IL-2 induced apoptosis, tissue sections from spleen and liver were analyzed for the number of apoptotic cells 6 days after the administration of Ag into IL-2-deficient mice reconstituted with IL-2 or PBS. As seen in Fig. 5, C and D, the number of apoptotic cells in the IL-2-treated mice was 2- to 10-fold higher than the number seen in mice given PBS. One IL-2-treated mouse did not exhibit an increase in apoptosis, which may be due to variability in the kinetics of the experiment, or perhaps due to an incomplete injection of IL-2. Nevertheless, these results suggest that IL-2, associated with heparan sulfate, is able to prime cells for apoptosis in vivo.

Because IL-2-deficient T cells may be abnormally responsive to exogenous IL-2, we asked whether heparan sulfate-bound IL-2 influences activation-induced cell death in normal T cells. To this end, TCR transgenic T cells specific for OVA (35) were transferred into nude mice, which exhibit no detectable extrafollicular IL-2, and established conditions wherein the T cells should undergo IL-2-dependent activation-induced cell death. Lenardo et al., have shown that IL-2-primed cell death occurs as a result of TCR reengagement of activated T cells during lymphokine-driven proliferation (36). We recapitulated these prerequisites by stimulating the OVA-specific T cells in vitro for 48 h, transferring the T cells into nude mice “reconstituted” with IL-2 or PBS, and then reimmunizing the nude mice with OVA given by i.p. injection. As seen in Fig. 5 E, the frequency of OVA-specific CD4⁺ T cells harvested from nude mice that had received IL-2 24 h before was decreased on day 5 compared with the frequencies in mice given PBS, consistent with our results in the IL-2 knockout mice. By day 10, the frequencies of OVA-specific T cells in the PBS and IL-2-treated mice were equal. Because the nude mice were infused with IL-2 competent cells (which reconstitute intrasplenic IL-2; data not shown), IL-2-mediated T cell loss was likely delayed but not abrogated in these animals.

We next asked whether heparan sulfate-bound IL-2 might induce proliferation in vivo. To this end, BALB/c nude mice were infused with IL-2 or PBS then injected 30 h later with IL-2-deficient, TCR transgenic T cells specific for OVA. Seventy-two hours later, the mice that had received the OVA-specific T cells were stimulated with 2 mg OVA. The T cell response was assessed 72 h later. This experiment excludes any possibility that brief exposure to soluble IL-2 might affect the unstimulated T cells, and tests how long IL-2 remains bound in functional form to heparan sulfate in vivo. As seen in Fig. 5 F, IL-2 deficient, OVA-specific T cells infused into mice with matrices that were reconstituted with IL-2 increased in frequency in response to OVA, whereas OVA-specific T cells in control nude mice did not. T cells isolated from peripheral (axillary/brachial/cervical) lymph nodes, perihepatic lymph nodes, and spleen all increased in frequency in response to IL-2, suggesting that this increase was due to proliferation and not due to differential homing. T cells transferred into nude mice receiving IL-2 but no Ag did not respond.

Our findings reveal a heretofore unknown localization of IL-2 by heparan sulfate in organs of the immune system. IL-2 so localized regulates T cell homeostasis by inducing proliferation and by priming cells for activation-induced cell death. Whether other properties of IL-2 such as enhancement of lymphocyte survival may be mediated by IL-2 while sequestered remains to be determined.

Because the studies of Ramsden and Rider (12) were strictly in vitro, it has been unclear to date what the functional outcome of an in vivo association between heparan sulfate and IL-2 might be. Scattered reports in the literature indirectly yield some insight regarding the functional implications of this interaction. Increased extracellular heparan sulfate within the spleen has been noted in an experimental myeloproliferative syndrome in mice (37). This syndrome, induced by a myeloproliferative sarcoma virus, causes significant splenomegaly. The authors postulated that the retention of cytokines by heparan sulfate may contribute to the cellular proliferation seen in this syndrome. Additionally, patients with certain lysosomal storage diseases resulting from an absence of enzymes necessary to degrade heparan sulfate exhibit hepatosplenomegaly. Through rodent models, we are currently investigating the potential role of heparan sulfate-bound IL-2 in these pathologies.

A variety of chemokines and cytokines bind heparan sulfate, and through this association local availability of these mediators is determined. In some cases, the binding of a cytokine to heparan sulfate modulates the bioactivity of that cytokine. For example, association of IL-8 with heparan sulfate enhances the chemotactic properties of IL-8 (4). This enhancement may occur through oligomerization of IL-8, which is induced with binding to heparan sulfate. Oligomerization of the chemokines RANTES, MIP-1α, and monocyte chemoattactant protein-1 (MCP-1) (38) is also induced via association with heparan sulfate. Binding to heparan sulfate influences the bioactivity of IFN-γ by protecting it from proteolytic degradation, and by increasing the on-rate of IFN-γ with its receptor (39). In the case of IL-2, we do not know as yet whether all IL-2 remains bound to heparan sulfate to exert its functions, or whether some IL-2 is released by enzymatic degradation of heparan sulfate. According to molecular modeling of the interaction between IL-2 and heparin, binding of IL-2 to heparin does not interfere with the binding of IL-2 to its receptor, leaving open the possibility that binding to heparan sulfate may alter the function of IL-2 (30). Because the IL-2R is composed of three chains (αβγ), with differing affinities for IL-2 depending on which combination of chains is expressed (βγ vs αβγ), the potential for altering the affinity of IL-2 for its receptor exists.

Previous work from this laboratory showed that heparan sulfate glycosaminoglycan activates APCs and through this means alters the development of immature T cells and moderates effector functions of mature T cells (40, 41, 42). These findings, in conjunction with our current studies, suggest that heparan sulfate has a profound influence on immune responses through modulation of both APC and T cell function. The genetic engineering of mice deficient in various enzymes requisite for the synthesis of heparan sulfate may yield further insights into this area.

Although it is not known at which site heparan sulfate-bound IL-2 has the greatest influence, IL-2 in the marginal zones and red pulp of the spleen is well positioned to regulate the survival of effector T cells produced via cognate interactions with exogenous or self Ags. In fact, under certain circumstances such as partial blockade of IL-2-heparan sulfate interactions with heparin or during reconstitution of IL-2-deficient mice with IL-2-producing cells, pronounced staining for IL-2 at the marginal zones, an area which is thought to be of import for lymphocyte trafficking to the spleen, is observed (our unpublished observations).

Given that heparan sulfate-bound IL-2 promotes proliferation and apoptosis in vivo and is localized predominantly in the spleen and thymus, our findings imply that splenectomized animals may experience some degree of immune dysregulation. Splenectomy has long been known to increase the risk of fatal bacterial sepsis, and splenectomized, immunosuppressed patients have an increased risk of cancer compared with immunosuppressed patients with an intact spleen (43, 44). In rodents, the response to allogeneic stimulation is accelerated and oral tolerance is abrogated by splenectomy (45, 46). Splenectomized humans exhibit increased numbers of lymphocytes and NK cells in their peripheral blood (47). Splenic atrophy and hyposplenism are associated with autoimmune-mediated gastrointestinal disorders and autoantibody production (48, 49). In IL-2-deficient mice with an intact spleen, autoimmunity in the form of autoimmune hemolytic anemia (through production of anti-RBC Abs) and colitis occurs. Anti-RBC autoantibodies may be produced as a result of defective elimination (via IL-2-primed apoptosis) of RBC-specific T cells arising in the spleen from the presentation of RBC Ags by macrophages. The production of anti-platelet autoantibodies in human patients treated with heparin (50) may likewise result from diminished levels of intrasplenic IL-2.

Several disease processes are accompanied by alterations in the amount/composition of heparan sulfate. Diabetes mellitus results in a decrease in heparan sulfate proteoglycan (51). Certain mucopolysaccharidoses and amyloidosis result in relative increases in tissue levels of heparan sulfate (52, 53). We would postulate that the immune dysregulation observed in these diseases might reflect, in part, the abnormal association of IL-2 and heparan sulfate. Therapeutic strategies aimed at modulating this association might prove fruitful as we gain information about the significance of this interaction for immune responses.

We thank Jeffrey Ansite, Peter Eckman, Dr. John Miller, Dr. Geoff Gersuk, and Michelle Heilman for expert technical assistance, and Dr. Andrew Farr for his critical review of the manuscript.