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The Journal of Immunology, 2001, 166: 188-196.
Copyright © 2001 by The American Association of Immunologists

Activated T Lymphocytes Regulate Hyaluronan Binding to Monocyte CD44 Via Production of IL-2 and IFN-{gamma}1

Marc C. Levesque2 and Barton F. Haynes

Department of Medicine, Division of Rheumatology, Allergy and Clinical Immunology, Department of Immunology, and Duke University Arthritis Center, Duke University Medical Center, Durham, NC 27710


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interactions of the cell surface proteoglycan CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA) are important during inflammatory immune responses. Our previous studies indicated that monocyte HA binding could be induced by TNF-{alpha}. Moreover, monocyte HA binding could be markedly up-regulated by culturing PBMC with anti-CD3 (TCR complex) mAbs. The present study was undertaken to identify soluble factors and/or cell surface molecules of activated T lymphocytes that might regulate HA binding to monocytes. Abs to IL-1{alpha}, IL-1{beta}, IL-2, IL-3, IL-10, IL-15, GM-CSF, IFN-{gamma}, and TNF-{alpha} were tested for their effects on anti-CD3 mAb-, Con A-, and PMA/ionomycin-mediated monocyte HA binding in PBMC cultures. Anti-TNF-{alpha}, anti-IL-2, and anti-IFN-{gamma} Abs, when added together to PBMC cultures, completely blocked Con A- and partially blocked anti-CD3- and PMA/ionomycin-induced monocyte HA binding. Furthermore, when added together to PBMC cultures, IL-2 and TNF-{alpha} induced high levels of monocyte HA binding. Likewise, IFN-{gamma} augmented TNF-{alpha}-induced monocyte HA binding. To investigate the role of T cell-monocyte direct contact in induction of monocyte HA binding, we studied PMA/ionomycin-activated, paraformaldehyde-fixed CD4+ T cells in these assays. Fixed, PMA/ionomycin-activated CD4+ T lymphocytes induced monocyte HA binding, but direct T cell-monocyte contact was not required. Moreover, anti-IFN-{gamma} and anti-TNF-{alpha} Abs blocked fixed PMA/ionomycin-activated CD4+ T cell-induced monocyte HA binding. Taken together, these studies indicate roles for soluble T lymphocyte-derived factor(s), such as IL-2 and IFN-{gamma}, and a role for monocyte-derived TNF-{alpha} in Con A-, TCR complex-, and PMA/ionomycin-induced HA binding to monocyte CD44.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interactions of the cell surface proteoglycan CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA)3 are important at multiple steps in the generation of inflammatory immune responses. In vivo, CD44 mAbs block both B and T cell activation (1), inhibit delayed-type hypersensitivity reactions (2), prevent Langerhans and dendritic cell emigration to lymph nodes after Ag uptake (3) and block T lymphocyte-endothelial cell rolling and emigration into inflammatory sites (4, 5). CD44 mAbs also block the development of joint swelling in both collagen- and proteoglycan-induced models of inflammatory arthritis in mice (6, 7).

Low molecular mass HA is an important mediator of chronic inflammation in rheumatoid arthritis (RA) through its effects on macrophage activation (8, 9, 10, 11, 12) and angiogenesis (13, 14, 15), both of which represent important components of synovial inflammation in RA (16). The proinflammatory effects of low molecular mass HA on macrophage activation and angiogenesis are mediated primarily by CD44 (9, 10, 17). CD44 expression is up-regulated in RA synovium (18, 19), and RA synoviocytes produce large quantities of low molecular mass HA (20, 21, 22). Low molecular mass HA (<1 x 106 kDa) induces macrophage activation as evidenced by macrophage proinflammatory cytokine production (8, 9, 10), NO production (11), and NF-{kappa}B activation (11, 12). Low molecular mass HA also induces endothelial cell proliferation (13, 14, 15). Thus, the ability to bind HA by monocytes and tissue macrophages is a key event in inflammation in RA.

Although most immune cell types express some form of CD44, not all immune cells constitutively bind HA (23). In particular, freshly isolated peripheral blood monocytes express abundant cell surface CD44 yet do not bind HA (18). Thus, binding of HA to CD44 is a highly regulated event. Our previous studies identified TNF-{alpha} as an important positive regulator, and IL-4 and IL-13 as important negative regulators, of monocyte HA binding in the absence of T cells (24). Others have demonstrated up-regulation of CD44 expression on B lymphocytes in response to IL-5 (25), and on airway smooth muscle cells (26), fibroblasts (27), HUVEC (28), and astrocytes (29) in response to TNF-{alpha}. Recently, Guo et al. (30) identified CD40 ligand-CD40 interactions as a regulator of CD44 expression on B cells.

Chronic inflammation is characterized by infiltration of tissues by activated T lymphocytes and macrophages (16). Furthermore, most models of chronic inflammation postulate a role for cytokines and T lymphocytes as critical regulators of macrophage activation (31, 32, 33). T lymphocytes may either regulate monocyte CD44 expression and HA binding through production of soluble factors or via cell surface interactions.

This study was undertaken to study induction of monocyte-HA binding by activated T cells and to identify the roles that soluble factors and/or direct cell contact of activated T lymphocytes and monocytes play in regulating HA binding to monocytes. We found that T cell-derived IL-2 and IFN-{gamma} in the absence of direct T cell contact with monocytes induced monocyte TNF-{alpha} release and high levels of monocyte HA binding.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

Anti-CD14-PE, IgG-FITC, and IgG2a-PE were obtained from Dako (Glostrup, Denmark). The anti-CD44 mAb 5F12 inhibits HA binding to CD44 (34). Neutralizing mAbs to human IL-1{beta} (mAb 8516.311), IL-2 (mAb 5334.21), IL-3 (mAb 4806.1), IL-10 (mAb 23738.11), IL-15 (mAb 34593.11), IFN-{gamma} (mAb 25718.11), and TNF-{alpha} (mAb 1825.121); goat polyclonal neutralizing Abs to IL-1{alpha}, IL-2, and GM-CSF; and the isotype control Abs IgG1 (mAb 11711.11), IgG2a (mAb 20102.1), and normal goat IgG were obtained from R&D Systems (Minneapolis, MN). Optimal concentrations (5 µg/ml or 10 µg/ml for mAbs and 10 µg/ml for goat polyclonal neutralizing Abs) of anti-cytokine neutralizing Abs were determined by titration experiments using wide concentration ranges (24). For some in vitro culture experiments, anti-IL-2, anti-IFN-{gamma}, and anti-TNF-{alpha} neutralizing Abs were used at a concentration of 50 µg/ml to improve the likelihood that IL-2, IFN-{gamma}, and TNF-{alpha}, respectively, were neutralized. No further effects on HA binding were seen with these increased doses of anti-IL-2, anti-IFN-{gamma}, and anti-TNF-{alpha} neutralizing Abs, compared with the lower doses of neutralizing Abs.

Cytokines and T lymphocyte mitogens

IL-2, IFN-{gamma}, and TNF-{alpha} were obtained from R&D Systems. LPS (Escherichia coli 055:B5), Con A, and PMA were obtained from Sigma (St. Louis, MO). PHA was obtained from Murex Diagnostics (Dartford, U.K.). Ionomycin was obtained from Calbiochem (La Jolla, CA). Anti-CD3 (mAb OKT3, cell line obtained from American Type Culture Collection, Manassas, VA) was used as supernatants from confluent hybridoma cultures. All T lymphocyte mitogens were titered to obtain maximal HA binding to monocytes in PBMC cultures and were used at the following concentrations: Con A at 0.5 µg/ml, anti-CD3 mAb at 1:80 dilution of culture supernatant, and PMA/ionomycin at 10 ng/ml and 0.5 µg/ml, respectively.

Flow cytometry and HA binding assay

HA binding to PB monocytes was assayed using saturating amounts of soluble HA-FITC in double-labeling protocols gating on CD14+ cells (35). HA-FITC (gift of Anika Therapeutics, Woburn, MA) was derived from rooster comb and had an average molecular mass of 6.9 x 105 Da. Unless otherwise stated, there were no subpopulations of monocytes with differential HA binding, i.e., analysis of HA binding to monocytes revealed uniform shifts in the entire monocyte population with cytokine and Ab treatments. Samples were analyzed on a FACStarPlus (Becton Dickinson, Mountain View, CA). The binding level of HA-FITC to freshly isolated or cultured monocytes that could not be blocked by unlabeled HA or anti-CD44 mAbs was considered nonspecific.

PBMC separation and cultures

PBMC from normal healthy donors were isolated by density centrifugation on Ficoll-Hypaque (36, 37). PBMCs (5 x 105, 2 x 106, or 4 x 106) were cultured for 72 h in 48-, 24-, and 12-well plates, respectively, in RPMI 1640 medium supplemented with 10% v/v autologous serum at 37°C, 5% CO2 in air. Autologous human serum was obtained simultaneously with peripheral blood and used in PBMC culture experiments. Cytokines and neutralizing Abs were added at the time of initiation of PBMC cultures.

Monocyte separation and cultures

Monocytes were purified from PBMC using a combination of magnetic beads for removal of lymphocytes and adherence to plastic (24). Briefly, PBMCs were treated with a mixture of magnetic beads coated with Abs to CD2 and CD19 (Dynal, Lake Success, NY) according to the manufacturer’s instructions. Further purification of monocytes was obtained by plating the remaining cells and removing nonadherent cells after culture for 2 h at 37°C. This procedure resulted in preparations of monocytes that were 92 ± 2.1% (mean ± SEM) (n = 22) monocytes as determined by nonspecific esterase staining. Monocytes (5 x 105) were cultured for 72 h in 48-well plates in RPMI 1640 medium supplemented with 10% v/v autologous serum at 37°C, 5% CO2 in air for HA binding assays (35). Autologous human serum was obtained simultaneously with peripheral blood and used in monocyte culture experiments. Cytokines and neutralizing Abs were added to monocyte cultures after removal of nonadherent cells.

CD4+ T lymphocyte separation

CD4+ T lymphocytes were separated from PBMCs using a combination of magnetic beads coated with an anti-CD4 mAb and CD4/CD8 Detachabeads to remove the cells from the beads as outlined by the manufacturer (Dynal). Briefly, 30–100 x 106 PBMC were incubated with 30 x 106 magnetic beads coated with an anti-CD4 mAb at 4°C for 1 h with rocking in 1 ml of RPMI 1640 medium plus 0.01% human serum albumin (Plasbumin-25; Bayer Corporation, Elkhart, IN). CD4+ cells were collected using a magnet, and nonadherent cells were removed. To improve purity, the magnetic beads and attached cells were gently resuspended in 3 ml of RPMI 1640 medium plus 0.01% human serum albumin, and CD4+ cells were again collected with a magnet, and nonadherent cells were removed. The magnetic beads and attached cells were resuspended in 100 µl of RPMI 1640 medium plus 10% autologous serum and 40 µl of CD4/CD8 Detachabeads, and the mixture was gently agitated at room temperature for 1 h. Detachment of cells from beads was monitored by examining the mixture under a microscope. Magnetic beads were removed with a magnet and nonadherent cells were collected, counted, pelleted by centrifugation at 400 x g for 5 min and resuspended at 1 x 107 cells/ml in RPMI 1640 medium. The purity of the preparations was monitored by flow cytometry and on average ± SEM were 94.8% ± 0.7% CD4+, 95.4% ± 0.7% CD3+, 2.3% ± 0.4 CD8+, 0.5% ± 0.2% CD20+, 1.8% ± 0.3% CD56+, and 0.2% ± 0.1% CD14+ (n = 22).

CD4+ T lymphocyte activation with PMA/ionomycin, paraformaldehyde-fixation, and culture

CD4+ T cells (1 x 106) were cultured in 0.5 ml of RPMI 1640 medium plus 10% autologous serum in 48-well plates with PMA (10 ng/ml) and ionomycin (0.5µg/ml) at 37°C, 5% CO2 in air for 0 to 72 h. Cells were counted and washed twice in PBS. Depending on the experiment, 2.5 x 105 to 1 x 106 CD4+ cells were fixed in 1 ml of 1% paraformaldehyde in PBS by gentle agitation at 4°C for 2 h. After fixation, the cells were washed twice in RPMI 1640 medium. For time course experiments, fixed, activated CD4+ lymphocytes were added to monocytes at a 1:1 ratio of lymphocytes to monocytes in 48-well plates in RPMI 1640 medium plus 10% autologous serum at 37°C, 5% CO2 in air for 72 h and harvested and assayed for HA binding as described earlier. In other experiments, the ratio of lymphocytes to monocytes was varied. To confirm the adequacy of the fixation protocol, CD4+ T lymphocytes were activated with PMA/ionomycin for 72 h and then tested in [3H]thymidine incorporation assays. Fixed cells, compared with unfixed cells, lacked the ability to incorporate [3H]thymidine.

In a series of experiments, T lymphocyte and monocyte coculture experiments were performed in 48-well Transwell culture devices (Costar, Cambridge, MA) in which the chambers were separated by a 0.4-µm polycarbonate membrane. Fixed PMA/ionomycin-activated CD4+ T lymphocytes were cultured in the upper chamber and monocytes in the lower chamber of the Transwell culture devices.

TNF-{alpha} and IFN-{gamma} supernatant ELISA

Measurement of TNF-{alpha} and IFN-{gamma} concentrations in the culture media of PMA/ionomycin-activated T lymphocytes was performed by ELISA according to the manufacturer’s instructions (Genzyme, Cambridge, MA and R&D Systems, respectively).

Statistical analysis

Paired Student’s t tests were used to compare the effects of cytokine neutralizing mAbs vs isotype-matched control mAbs, the effects of IL-2, TNF-{alpha}, and IFN-{gamma}, and the effects of direct contact vs separation by a permeable membrane of fixed PMA/ionomycin-treated CD4+ T cells on monocyte HA binding.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of PHA, Con A, anti-CD3 mAb, or PMA/ionomycin on HA binding to monocytes in PBMC cultures and in cultures of purified monocytes

PHA and anti-CD3 mAb treatment of PBMC cultures induces increased HA binding to monocytes (18). Because it is possible that PHA and anti-CD3 mAb act directly on monocytes rather than via lymphocytes to induce monocyte HA binding, we performed HA binding assays on purified monocytes treated with anti-CD3 mAb or PHA. PHA, but not anti-CD3 mAb, induced HA binding to purified monocytes (Fig. 1GoA). Because PHA contained endotoxin (0.3 EU/ml {cong} 0.03 ng/ml at 0.5 µg/ml PHA), further studies with this reagent were not pursued. Two other T cell mitogens, Con A and PMA/ionomycin, also induced monocyte HA binding in PBMC cultures and Con A and the anti-CD3 mAb contained <0.01 EU/ml of endotoxin (Fig. 1GoA). Like anti-CD3, neither Con A nor PMA/ionomycin induced HA binding to purified monocytes. Importantly, 81, 91, and 94% HA binding to monocytes in anti-CD3-, Con A-, and PMA/ionomycin-treated PBMC cultures, respectively, could be blocked with the anti-CD44 mAb 5F12 (Fig. 1GoB).



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  		FIGURE 1. Effect of PHA, Con A, anti-CD3 mAb, and PMA/ionomycin on HA binding to monocytes in PBMC cultures and purified monocytes. PBMC (A and B) or purified monocytes (A) were isolated and cultured in RPMI 1640 medium plus 10% autologous serum containing no mitogen, PHA (0.5 µg/ml), anti-CD3 (1:80 dilution of culture supernatant), Con A (0.5 µg/ml), or PMA/ionomycin (10 ng/ml and 0.5 µg/ml, respectively) for 72 h. Monocyte {Delta}MFI values for HA binding to CD14+ cells were determined as follows: {Delta}MFI = (MFI of HA-FITC binding to CD14+ cells) - (MFI of IgG-FITC binding to CD14+ cells). In B, harvested PBMCs were preincubated with the anti-CD44 mAb 5F12 or a control isotype-matched Ab before staining with HA-FITC. Data represent the mean ± SEM of three experiments. In A, p values for HA binding to monocytes in no mitogen, PHA-, anti-CD3-, Con A-, and PMA/ionomycin-stimulated monocyte cultures vs PBMC cultures were 0.783, 0.638, 0.118, 0.075, and 0.042, respectively. In B, p values for monocyte HA binding assays performed in the presence of a control mAb vs anti-CD44 mAb in anti-CD3-, Con A-, and PMA/ionomycin-stimulated PBMC cultures were 0.001, 0.011, and 0.072, respectively.

 
Cytokine regulation of HA binding to PBMC monocyte CD44 induced by Con A, anti-CD3 mAb, PMA/ionomycin, or LPS

Neutralizing Abs to cytokines previously identified as inducers of HA binding to monocytes (IL-1{alpha}, IL-1{beta}, IL-2, IL-3, IL-10, IL-15, GM-CSF, and TNF-{alpha}) (24) and IFN-{gamma} were tested for their effects on anti-CD3-, Con A-, and PMA/ionomycin-mediated HA binding in PBMC cultures. For comparison, similar studies were performed with LPS, which acts directly on monocytes to induce HA binding (24) (Table IGo). A neutralizing Ab to TNF-{alpha} significantly inhibited 43% (p < 0.0001), 74% (p = 0.0002), and 24% (p = 0.005) of monocyte HA binding induced by anti-CD3, Con A, and, as reported, LPS (24), respectively. In addition, Con A-induced HA binding to monocytes was significantly inhibited by 38% (p = 0.0081) by a neutralizing Ab to IL-2 and by 42% (p = 0.0357) by a neutralizing mAb to IFN-{gamma}.


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  		Table I. Effect of anti-cytokine Abs on monocyte CD44 HA binding mediated by Con A, anti-CD3 mAb, PMA/ionomycin, or LPS

 
Interestingly, under the conditions tested in Table IGo, in contrast to anti-CD3- and Con A-stimulated PBMC, PMA/ionomycin-induced HA binding to monocytes in PBMC cultures was not blocked by a neutralizing anti-TNF-{alpha} mAb. Therefore, supernatants from PMA/ionomycin-treated PBMC cultures were assayed for TNF-{alpha} and found to contain 15 ng/ml of TNF-{alpha} (Fig. 2Go). Titration of the ionomycin concentration in PMA/ionomycin-stimulated PBMC cultures resulted in decreases in the TNF-{alpha} concentration in these cultures and resulted in partial inhibition of PMA/ionomycin-induced HA binding in PBMC cultures by an anti-TNF-{alpha} neutralizing Ab (Fig. 2Go). Thus, taken together, the data in Table IGo and Fig. 2Go suggested that TNF-{alpha} was involved in the induction of HA binding to monocytes in anti-CD3-, PMA/ionomycin-, and Con A-stimulated PBMC cultures.



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  		FIGURE 2. Effect of a TNF-{alpha} neutralizing Ab on HA binding to monocytes in PBMC cultures treated with PMA and various concentrations of ionomycin and measurement of the TNF-{alpha} concentration produced in these cultures. PBMCs were isolated and cultured for 72 h with PMA (10 ng/ml) and various concentrations of ionomycin (0.5, 0.05, 0.005, and 0 µg/ml) with an anti-TNF-{alpha} neutralizing mAb (5µg/ml) or an isotype-matched control mAb. Monocyte {Delta}MFI values, for HA binding to CD14+ cells, were determined as described in Fig. 1Go. The TNF-{alpha} concentration in the culture media was determined by ELISA. Data represent the mean ± SEM of three experiments. Values of p for IgG1 {Delta}MFI vs anti-TNF-{alpha} {Delta}MFI for PMA (ng/ml)/ionomycin (µg/ml) concentrations of 10/0.5, 10/0.05, 10/0.005, and 10/0 were 0.125, 0.009, 0.058, and 0.087, respectively.

 
Various combinations of IL-2, TNF-{alpha}, and IFN-{gamma} neutralizing Abs were tested for their ability to inhibit monocyte HA binding of PBMC treated with Con A or anti-CD3. We found complete inhibition of HA binding to monocytes in Con A-stimulated PBMC cultures by a combination of IL-2, TNF-{alpha}, and IFN-{gamma} neutralizing Abs (Fig. 3Go). However, only anti-TNF-{alpha} partially inhibited anti-CD3-induced HA binding to monocytes (Fig. 3Go).



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  		FIGURE 3. Effect of IFN-{gamma}, IL-2, and TNF-{alpha} neutralizing Abs on HA binding to monocytes in PBMC cultures treated with either Con A or anti-CD3 mAb. PBMC were isolated and cultured for 72 h with Con A or anti-CD3 mAb as described in Fig. 1Go. Monocyte {Delta}MFI values, for HA binding to CD14+ cells, were determined as described in Fig. 1Go. Data represent the mean ± SEM. For Con A-treated cultures, p < 0.05 for all neutralizing mAb vs control mAb treatments, p < 0.05 for anti-IL-2 vs anti IL-2/anti-TNF-{alpha}, anti-IL-2 vs anti-IL-2/anti-TNF-{alpha}/anti-IFN-{gamma}, anti-TNF-{alpha} vs anti-IL-2/anti-TNF-{alpha}, anti-IFN-{gamma} vs anti-IFN-{gamma}/anti-TNF-{alpha}, and anti-IFN-{gamma} vs anti-IL-2/anti-TNF-{alpha}/anti-IFN-{gamma}, and p = NS for all other comparisons. For anti-CD3-treated cultures p < 0.05 for all neutralizing mAb vs control mAb treatments except anti-IL-2 vs control mAb and anti-IL-2/anti-IFN-{gamma} vs control mAb and p < 0.05 for anti-IL-2 vs anti-IL-2/anti-IFN-{gamma}, anti-IL-2 vs anti-IL-2/anti-TNF-{alpha}/anti-IFN-{gamma}, anti-TNF-{alpha} vs anti-TNF-{alpha}/anti-IFN-{gamma}, anti-IFN-{gamma} vs anti-TNF-{alpha}/anti-IFN-{gamma}, anti-IFN-{gamma} vs anti-IL-2/anti-TNF-{alpha}/anti-IFN-{gamma}, and p = NS for all other comparisons.

 
Effect of IL-2 and TNF-{alpha} on HA binding to monocytes in PBMC cultures and in purified suspensions of monocytes

IL-2 and TNF-{alpha} were added both alone and together to PBMC and purified monocyte cultures to determine the effects of a combination of these cytokines on HA binding to monocytes. As shown in Fig. 4GoA, IL-2 and TNF-{alpha}, when added together, induced high-level HA binding to monocytes in PBMC cultures, compared with lower levels of HA binding induced by either cytokine alone (p < 0.05 for comparison of {Delta} mean fluorescence intensity (MFI) values for HA binding for IL-2 and TNF-{alpha} together vs either cytokine alone at all concentrations tested). In contrast, coculture of purified monocytes with IL-2 and TNF-{alpha} together did not result in augmented HA binding, compared with culture of purified monocytes in TNF-{alpha} or IL-2 alone (p = NS) (Fig. 4GoB). As shown previously (24), coculture of purified monocytes with IL-2 alone did not augment HA binding (Fig. 4GoB). These results suggested that in this experimental design, monocyte TNF-{alpha} was the primary soluble mediator of mitogen-induced PBMC monocyte binding to HA and that IL-2 acted on peripheral blood T cells to indirectly mediate monocyte HA binding.



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  		FIGURE 4. Effect of IL-2 and TNF-{alpha} on HA binding to monocytes in PBMC cultures and to purified monocytes. PBMC (A) and purified monocytes (B) were isolated and cultured for 72 h with either no cytokine or IL-2 (10 ng/ml) and various concentrations of TNF-{alpha} (0, 0.1, 1, or 10 ng/ml). Monocyte {Delta}MFI values, for HA binding to CD14+ cells, were determined as described in Fig. 1Go. Data represent the mean ± SEM of three experiments, except IL-2 only treatment (n = 8) and TNF-{alpha} only treatment at 10 ng/ml (n = 6). For PBMC cultures (A), p < 0.05 for comparison of {Delta}MFI values for HA binding for IL-2 and TNF-{alpha} together vs either cytokine alone at all concentrations tested. For monocyte cultures (B), p = NS for comparison of {Delta}MFI values for HA binding for IL-2 and TNF-{alpha} together vs either cytokine alone at all concentrations tested.

 
Effect of IFN-{gamma} and TNF-{alpha} on HA binding to monocytes in PBMC cultures and in purified suspensions of monocytes

IFN-{gamma} and TNF-{alpha} were added both alone and together to PBMC and purified monocyte cultures to determine the effects of a combination of these cytokines on HA binding to monocytes. As shown in Fig. 5Go, IFN-{gamma} and TNF-{alpha}, when added together, induced high-level HA binding to monocytes in PBMC cultures and to purified monocytes, compared with lower levels of HA binding induced by either cytokine alone. Higher concentrations of IFN-{gamma} (100 and 1000 U/ml) augmented monocyte HA binding in purified monocyte cultures without TNF-{alpha} but did not further augment exogenous TNF-{alpha}-induced monocyte HA binding in these cultures, compared with 10 U/ml of IFN-{gamma} (Fig. 5Go, C and D). In addition, IFN-{gamma}-induced monocyte HA binding was blocked by an anti-TNF-{alpha}-neutralizing Ab (74 ± 6.7% (mean ± SEM) inhibition of HA binding for comparison of monocyte HA-binding {Delta}MFI in cultures with IgG1 vs anti-TNF-{alpha} (n = 3, p = 0.04)). Therefore, the ability of IFN-{gamma} to induce HA binding to monocytes did not require T cells but did require monocyte-derived TNF-{alpha}.



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  		FIGURE 5. Effect of IFN-{gamma} and TNF-{alpha} on HA binding to purified monocytes. Purified monocytes were isolated and cultured for 72 h with either no cytokine or IFN-{gamma} (10 U/ml) and various concentrations of TNF-{alpha} (0, 0.1, 1, or 10 ng/ml) (A and B) and with either no cytokine or TNF-{alpha} (1 ng/ml) and various concentrations of IFN-{gamma} (0, 10, 100, and 1000 U/ml) (C and D). Monocyte {Delta}MFI values, for HA binding to CD14+ cells, were determined as described in Fig. 1Go. Data represent the mean ± SEM of three experiments. For A, p = NS for comparison of {Delta}MFI values for HA binding for IFN-{gamma} and TNF-{alpha} together vs either cytokine alone at all concentrations tested except for comparison of IFN-{gamma} only vs IFN-{gamma} and TNF-{alpha} (1 ng/ml), p = 0.02. For B, p < 0.05 for comparison of {Delta}MFI values for HA binding for IFN-{gamma} and TNF-{alpha} together vs cytokine alone at all concentrations tested except for comparison of IFN-{gamma} and TNF-{alpha} (0.1 ng/ml) vs either cytokine alone, p = NS. For C, p = NS for comparison of {Delta}MFI values for HA binding for IFN-{gamma} and TNF-{alpha} together vs TNF-{alpha} alone at all concentrations tested and p < 0.05 for comparison of {Delta}MFI values for HA binding for IFN-{gamma} and TNF-{alpha} together vs IFN-{gamma} alone at all concentrations tested. For D, p < 0.05 for comparison of {Delta}MFI values for HA binding for IFN-{gamma} and TNF-{alpha} together vs cytokine alone at all concentrations tested except for comparison of IFN-{gamma} (100 U/ml) and TNF-{alpha} vs cytokine alone, p = NS.

 
HA binding to monocytes induced by fixed PMA/ionomycin-activated CD4+ T lymphocytes

The studies presented in Table IGo and Figs. 2Go and 3Go suggested that T cell-produced TNF-{alpha} and IFN-{gamma} or direct contact of monocytes with T cells or both were involved in anti-CD3- and PMA/ionomycin-induced HA binding to monocyte CD44. To directly test the hypothesis that direct contact of monocytes with T cells may be involved in inducing monocytes to bind HA, purified CD4+ T lymphocytes were activated with PMA/ionomycin for various lengths of time, then fixed in 1% paraformaldehyde, washed extensively, and added to purified monocyte suspensions. T cell fixation with 1% paraformaldehyde has been used by other investigators to study the role of T cell surface molecules on macrophage activation (38). A summary of this experimental design is presented in Fig. 6Go; experimental results are presented in Fig. 7Go. We found that coculture of purified monocytes with fixed PMA/ionomycin-treated CD4+ T lymphocytes induced HA binding to monocyte CD44. The monocyte HA binding mediated by PMA/ionomycin-treated CD4+ T lymphocytes began within 2 h of addition of PMA/ionomycin to CD4+ T cells, peaked in 24 h after PMA/ionomycin addition, and began to diminish by 72 h (Fig. 7GoA).



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  		FIGURE 6. Experimental design to test postulate that CD4+ T lymphocyte cell surface molecules induce monocyte HA binding. CD4+ T lymphocytes were isolated with anti-CD4 magnetic beads and detached from the beads as described in Materials and Methods. PMA (10 ng/ml) and ionomycin (0.5 µg/ml) were added to the CD4+ T cells at various time points over the next 72 h. PMA/ionomycin-treated CD4+ T lymphocytes were washed in PBS, fixed in PBS containing 1% paraformaldehyde, and then washed in RPMI 1640 medium. Fixed PMA/ionomycin-activated CD4+ T cells were then counted and added in culture at a 1:1 ratio (T cells:monocytes) to unfixed monocytes for 72 h. Monocyte {Delta}MFI values, for HA binding to CD14+ cells, were determined as described in Fig. 1Go.

 


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  		FIGURE 7. Time course and T cell:monocyte ratio analysis of the effect of PMA/ionomycin-treated, fixed CD4+ T cells on monocyte HA binding. A, Monocyte {Delta}MFI values, for HA binding to CD14+ cells, were determined as described in Fig. 6Go. Data represent the mean ± SEM of three experiments. B, CD4+ T cells were isolated and activated for 72 h with PMA/ionomycin and then fixed, counted, and added to monocytes in the ratios shown. Monocyte {Delta}MFI values, for HA binding to CD14+ cells, were determined as described in Fig. 1Go. Data represent the mean ± SEM of three experiments.

 
To define the optimal culture conditions for fixed PMA/ionomycin-treated CD4+ T cells to induce HA binding to monocytes, we defined the optimal ratio of T lymphocytes to monocytes necessary to induce HA binding to monocytes (Fig. 7GoB). T cells activated with PMA/ionomycin for 72 h, optimally stimulated monocytes to bind HA at T cell:monocyte ratios of ~5:1 or greater. Paraformaldehyde-fixed CD4+ T cells activated with anti-CD3 could also induce HA binding to monocytes but required higher ratios of CD4+ lymphocytes to monocytes (8:1) to achieve a similar effect on HA binding to monocytes, compared with PMA/ionomycin-treated CD4+ T cells (data not shown).

Role of soluble factors produced by fixed PMA/ionomycin-treated CD4+ T cells on monocyte CD44 HA binding

Previous studies have suggested that after fixation with 1% paraformaldehyde that PMA/ionomycin-activated T cells can "leak" cytokines (39). To test this possibility, CD4+ T cells were treated with PMA/ionomycin for various amounts of times as diagramed in Fig. 6Go, fixed, and then added either directly to purified monocytes or added to the upper chamber of a Transwell culture device with purified monocytes in the lower chamber separated from the T cells by a 0.4-µm polycarbonate membrane. As shown in Fig. 8Go, fixed PMA/ionomycin-treated T cells induced nearly identical amounts of monocyte HA binding regardless of whether T cells were in direct contact with monocytes or were separated from monocytes by a Transwell culture device. Thus, these studies suggested that soluble factors and not direct T cell contact with monocytes were the primary mediators of HA binding to monocyte CD44.



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  		FIGURE 8. Comparison of monocyte HA binding induced by PMA/ionomycin-activated and fixed CD4+ T cells when the two cell types are cocultured vs separated by a 0.4-µm polycarbonate membrane. CD4+ T cells were isolated, PMA/ionomycin-activated for various times (0–72 h), and fixed with 1% paraformaldehyde in PBS as described in Fig. 6Go and either cocultured with monocytes or added to the upper chambers of Transwell culture devices separated from the lower monocyte containing chamber by a 0.4-µm polycarbonate membrane for 72 h. Monocyte {Delta}MFI values, for HA binding to CD14+ cells, were determined as described in Fig. 1Go. Data represent the mean ± SEM of three experiments.

 
Role of IL-2, IFN-{gamma}, and TNF-{alpha} in monocyte HA binding induced by fixed PMA/ionomycin-activated T cells

Next, we tested the effects of coculture of anti-IL-2, anti-IFN-{gamma}, and anti-TNF-{alpha} neutralizing Abs on CD4+ T cell-monocyte cultures in which T cells had been previously treated with PMA/ionomycin for 24 h and fixed with 1% paraformaldehyde. As shown in Fig. 9Go, anti-IFN-{gamma}- and anti-TNF-{alpha}-neutralizing Abs, but not anti-IL-2-neutralizing Ab, resulted in blockade of monocyte HA binding induced by fixed, PMA/ionomycin-activated CD4+ T cells.



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  		FIGURE 9. Effect of anti-IL-2, anti-IFN-{gamma}, and anti-TNF-{alpha} neutralizing Abs on PMA/ionomycin-activated CD4+ T lymphocyte-induced monocyte HA binding. CD4+ T cells were isolated, PMA/ionomycin-activated for 24 h and fixed with 1% paraformaldehyde in PBS and counted as described in Fig. PMA-activated, and fixed CD4+ T cells (6.5 x 105) were cocultured with autologous monocytes at a T cell:monocyte ratio of 1:1 for 72 h in media containing either a control isotype-matched Ab (IgG1 for anti-TNF-{alpha} and anti-IL-2 and IgG2a for anti-IFN-{gamma}) or an anti-IL-2, anti-IFN-{gamma}, or anti-TNF-{alpha} neutralizing Ab. Monocyte {Delta}MFI values, for HA binding to CD14+ cells, were determined as described in Fig. 1Go. Data represent the mean ± SEM of four experiments. p = 0.01 and p = 0.07 for HA binding for IgG1 vs anti-TNF-{alpha} and anti-IL-2, respectively. p = NS for IgG2a vs anti-IFN-{gamma}.

 
Measurement of IFN-{gamma} and TNF-{alpha} released from fixed PMA/ionomycin-treated CD4+ T lymphocytes

We measured the concentration of IFN-{gamma} by ELISA in the supernatants of PMA/ionomycin-treated CD4+ T cells cultured for 24 h both before and after fixation. Supernatants from CD4+ T cells treated with PMA/ionomycin for 24 h contained >7 ng IFN-{gamma}/1 x 106 cells (n = 3). By comparison, supernatants generated by incubation of these same cells after fixation with paraformaldehyde for another 24 h in RPMI 1640 medium contained 0.046 ± 0.017 ng (~0.5–1 U/ml) IFN-{gamma}/1 x 106 cells (n = 3).

Finally, we measured TNF-{alpha} levels by ELISA in the supernatants of fixed PMA/ionomycin-treated CD4+ T cells cultured for 24 h both before and after fixation. Supernatants from CD4+ T cells treated with PMA/ionomycin for 24 h contained >2.5 ng TNF-{alpha}/1 x 106 cells (n = 3). By comparison, supernatants generated by incubation of these same cells after fixation for another 24 h in RPMI 1640 medium contained 0.004 ± 0.001 ng/ml TNF-{alpha}/1 x 106 cells (n = 3). As shown in Figs. 4Go and 5Go, by itself, rTNF-{alpha} induced monocyte HA binding only at concentrations approximately >1 ng/ml and even in combination with IL-2 or IFN-{gamma}, rTNF-{alpha} induced only low-level HA binding at 0.1 ng/ml.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we found that anti-TNF-{alpha}, anti-IL-2, and anti-IFN-{gamma} neutralizing Abs completely blocked Con A-induced monocyte HA binding in PBMC cultures. Together, IL-2 and TNF-{alpha} induced synergistic monocyte HA binding in PBMC cultures but not in purified monocyte cultures, and low concentrations of IFN-{gamma} (10 U/ml) enhanced TNF-{alpha}-induced monocyte HA binding. We also found no significant role for T cell surface direct contact with monocytes to induce HA binding. Taken together, our studies indicate a role for soluble T cell-derived factor(s) (IL-2, IFN-{gamma}, and TNF-{alpha}) and a role for monocyte-derived TNF-{alpha} in regulating HA binding to monocyte CD44 (Fig. 10Go).



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  		FIGURE 10. Model of activated T lymphocyte regulation of HA binding to monocyte CD44.

 
Our studies with anti-cytokine neutralizing Abs identified differences between Con A-, anti-CD3 mAb-, and PMA/ionomycin-induced monocyte HA binding. Other studies noted differences between Con A- and anti-CD3 mAb-induced T cell IFN-{gamma} production and the ability of an anti-IL-2R mAb to inhibit IFN-{gamma} production in that an anti-IL-2R mAb completely inhibited Con A-induced but not anti-CD3 mAb-induced T cell IFN-{gamma} production (40). Thus, we suggest that the differences that we found in our experiments with anti-cytokine neutralizing Abs between Con A-, anti-CD3-, and PMA/ionomycin-induced monocyte HA binding were likely due to differences in the relative amounts of IL-2, IFN-{gamma}, and TNF-{alpha} produced by these mitogens. Anti-CD3/TCR-triggered T cell activation and monocyte HA binding is most physiologic, but there are significant differences between anti-CD3 mAb- and Ag-induced T cell activation with regard to the duration and strength of signals delivered to the T cell (41).

Several possible explanations exist to explain the inability of anti-TNF-{alpha} neutralizing Abs to completely block PMA/ionomycin-or anti-CD3 mAb-induced monocyte HA binding in whole PBMC cultures. It is possible that several cytokines may be involved in PMA/ionomycin- or anti-CD3 mAb-induced monocyte HA binding; the effects of TNF-{alpha} would, therefore, be redundant and neutralization of only TNF-{alpha} in the PMA/ionomycin- or anti-CD3 mAb-activated PBMC cultures would not be sufficient to completely block monocyte HA binding. A second possibility is that the large amount of TNF-{alpha} made by PMA/ionomycin- or anti-CD3 mAb-activated T cells was not effectively neutralized, although the concentration of anti-TNF-{alpha} neutralizing Ab used in these studies was titered to neutralize 50 ng/ml of rTNF-{alpha}.

Our experiments using fixed PMA/ionomycin-activated T cells were designed to determine whether cell surface interactions between T cells and monocytes induce monocyte CD44 HA binding. These experiments suggested that T cell-monocyte cell surface interactions were not necessary to induce monocyte HA binding and that soluble factors, including IFN-{gamma} and TNF-{alpha}, are important regulators of monocyte HA binding. Of importance, these experiments did not preclude the possibility that other soluble factors were involved in T cell-induced monocyte HA binding.

One of the key soluble factors released by activated T cells that induced monocyte HA binding seems to be IFN-{gamma}. A recent study by Weiss et al. (42) suggested that IFN-{gamma} regulates CD44 HA binding to monocytes. IFN-{gamma} is rapidly produced upon T cell activation with a time course comparable with that seen in Fig. 7Go for fixed PMA/ionomycin-induced monocyte HA binding (43). The inability of an anti-IFN-{gamma} neutralizing Ab to block anti-CD3- and PMA/ionomycin-induced monocyte HA binding is likely due to the effects of other cytokines, such as IL-2 and TNF-{alpha}, that are produced by activated T cells. Therefore, these studies suggest that in the setting of activated PBMC cultures, TNF-{alpha} is necessary for induction of monocyte HA binding and its production is either directly or indirectly augmented by IL-2 and IFN-{gamma} and perhaps other cytokines.

In summary, the studies in this article have identified monocyte TNF-{alpha} as an important mediator via T cell produced cytokines (such as IL-2 and IFN-{gamma}) of activated T lymphocyte-induced HA binding to monocyte CD44. Moreover, our studies do not support a requirement for direct contact of T cells with monocytes for induction of monocyte HA binding.


   Acknowledgments
 
We thank Dr. Larry Liao for insightful discussions, Mary Misukonis for performance of nonspecific esterase stains, and Erlina Siragusa and Joe Horvatinovich of the Center for AIDS Research Cell Sorter Facility for flow cytometry expertise.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AR01918-02 and AR39162. Back

2 Address correspondence and reprint requests to Dr. Marc C. Levesque, Durham Veterans Affairs and Duke University Medical Centers, 508 Fulton Street, Box 151-G, Durham, NC 27705. Back

3 Abbreviations used in this paper: HA, hyaluronan; RA, rheumatoid arthritis; MFI, mean fluorescence intensity. Back

Received for publication April 21, 2000. Accepted for publication October 2, 2000.


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 Results
 Discussion
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