Thrombin Receptor Expression and Responsiveness of Human Monocytic Cells to Thrombin Is Linked to Interferon-Induced Cellular Differentiation

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1 JOURNAL OF CELLULAR PHYSIOLOGY 177:76 84 (1998) Thrombin Receptor Expression and Responsiveness of Human Monocytic Cells to Thrombin Is Linked to Interferon-Induced Cellular Differentiation ANTONELLA NALDINI, 1 * LAURIE SOWER, 2 VELIO BOCCI, 1 BECKY MEYERS, 2 AND DARRELL H. CARNEY 2 1 University of Siena, Institute of General Physiology, Siena, Italy 2 Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas Human thrombin has been shown to stimulate monocyte chemotaxis, phagocytosis, and interleukin (IL8) production, but the mechanisms responsible for stimulation are not well defined. In some cells, thrombin stimulation of proliferation appears to require both cleavage of the proteolytically activated receptor for thrombin (PAR1) and activation of a nonproteolytically activated thrombin receptor (N-PAR), while in others activation of either receptor alone may be sufficient for stimulation. We, therefore, have initiated studies to address thrombin receptor expression and cell responsiveness to thrombin in interferon gamma (IFNg)-differentiated and nondifferentiated U937 monocytic cells. Northern blot analysis shows that PAR1 expression is upregulated upon differentiation. Experiments with biotinylated and 125 I-thrombin show that specific thrombin binding is dramatically increased by differentiation although it is not clear if this binding is to PAR1 or to a separate binding component such as N-PAR which is present on fibroblasts and other cells. Addition of thrombin at concentrations of 1 10 mg/ml ( nm, concentrations where specific thrombin binding is observed) stimulates proliferation of IFNg-differentiated U937 cells but not of undifferentiated U937 cells. Thrombin also stimulates interleukin-6 (IL6) production in IFNg-differentiated U937 cells. Moreover, thrombin induces high levels of IL6, interleukin-1b (IL1b), and tumor necrosis factor-a (TNFa) production by peripheral blood mononuclear cells (PBMC) and monocytes. These results show that differentiated U937 cells and mature PBMC are responsive to thrombin whereas nondifferentiated U937 are not. Further, this responsiveness appears to correlate with expression of PAR1 and to a dramatic increase in specific thrombin binding. That thrombin stimulates cytokine production and proliferation in populations of differentiated monocytes suggests that thrombin may be an important regulator of inflammation and wound healing. J. Cell. Physiol. 177:76 84, Wiley-Liss, Inc. In addition to its pivotal role in coagulation, throm- phagocytic activity (Sower et al., 1996). Thrombin also bin interacts with many types of cells to induce a varilogic may affect other aspects of inflammation and immuno- ety of cellular functions. Thrombin stimulates prolifercytokine surveillance by stimulating T-cell activation and ation of fibroblasts (Carney and Cunningham, 1978), production (Naldini et al., 1993) and modulaation endothelial cells (Gospodarowicz et al., 1978), neuronal tion of both natural killer (NK) and lymphokine-acticells (Gurwitz and Cunningham, 1988), vascular vated killer (LAK) cell cytotoxicity (Naldini and Car- smooth cells (Weiss and Nuccitelli, 1992), and bone ney, 1996). marrow cells in the presence of colony stimulating fachave Many of these cellular events induced by thrombin tor-1 (CSF-1; Clohisy et al., 1990). Further, thrombin been shown to require both proteolytic activation has potential to affect local inflammatory response by acting as a chemotactic agent for polymorphonuclear cells (PMNs) and monocytes (Bar-Shavit et al., 1983; Contract grant sponsor: M.U.R.S.T.; Contract grant sponsor: Consorzio Bizios et al., 1986) by stimulating the production of Siena Ricerche Fund; Contract grant sponsor: NIH; Con- monocyte chemotactic protein-1 (MCP-1; Colotta et al., tract grant number: GM ), by stimulating proliferation of monocytes (Cloh- *Correspondence to: Dr. Antonella Naldini, Institute of General isy et al., 1990), by stimulating the release of interleu- Physiology, University of Siena, Via Laterina 8, Siena, kin-1 (IL1) and interleukin-8 (IL8; Jones and Geczy, Italy ; Sower et al., 1996), and by enhancing monocyte Received 5 December 1997; Accepted 20 February WILEY-LISS, INC.

2 THROMBIN-INDUCED MONOCYTE ACTIVATION 77 of the G-protein-linked proteolytically activated recep- composition of cells in the suspension was Ç90% T lymphocytes tor for thrombin (PAR1) and binding to or activation (CD3/), Ç10% NK cells (CD16/), 0% monotor of a separate nonproteolytically activated receptor (N- cytes (CD14/), and 0% B lymphocytes (CD19/) asas- PAR) (Carney et al., 1986; Grand et al., 1996). It has sessed by flow cytometry. Monocyte-enriched popula- been reported that the majority of NK and T cells and tions were obtained by plating PBMC ( cells monocytes express PAR1 (Kudahl et al., 1991; Tordai per well) in 200 ml medium in 96-well tissue culture et al., 1993). In certain fibroblasts and other cells, how- microplates and allowed to adhere for 2 hr at 37 C. ever, activation of PAR1 is not sufficient to stimulate Nonadherent cells were removed by washing eight cell proliferation (Bar-Shavit et al., 1986; Brass et al., times with phosphate-buffered saline (PBS). Fresh serum-free 1991; Vouret-Craviari et al., 1992). Interestingly, certain medium (100 ml/well) was added and cells cells including neutrophils, bind thrombin with were cultured for the indicated intervals as described moderate/ high affinity (Kd ÅÇ2 nm), yet they do not below. Neutral red phagocytosis assays revealed that express PAR1 (Bizios et al., 1986). In these neutrophils, 95% of the adherent cells were monocytes (Gardner and thrombin stimulates neutrophil chemotaxis by a mechanism Remington, 1978). that appears to be independent of proteolytic activity or activation of PAR1 (Jenkins et al. 1995). We, U937 cell culture and IFNg treatment therefore, wanted to determine whether expression of U937 cells were cultured in RPMI-1640 supplemented PAR1, N-PAR, or both correlated with thrombin stimulation with 10% heat-inactivated FCS, 2 mm glutamine, 100 of monocyte proliferation and cytokine produc- IU/ml penicillin, and 100 mg/ml streptomycin. Cells were tion. For these studies, we compared PAR1 expression, induced to differentiate by supplementing the medium thrombin binding, and cell responsiveness in IFNg-dif- with 200 IU/ml IFNg for 10 days as previously described ferentiated and undifferentiated human monocytic (Roberts et al., 1991). Every 2 days the cells were resus- U937 cells derived from a histiocytic lymphoma (Sunds- pended in fresh medium containing IFNg to maintain tröm and Nilsson, 1976). It has been shown that IFNg, the cell concentration between cells/ml. After which is an important macrophage activating factor, 10 days, cells were washed extensively and cultured as induces changes in the U937 cell line that reflect cellular described below. To determine whether IFNg treatment differentiation (Ucla et al., 1990; Roberts et al., induced differentiation in U937 cells, cell proliferation 1991). Therefore, U937 cells provide a convenient was routinely tested, showing a decrease of about 50% model system to assess thrombin responsiveness dur- than controls as previously described (Oberg et al., 1991). ing monocyte differentiation. These studies show that Moreover, U937 cells were analyzed by a Nitro Blue Tet- thrombin induces proliferation and cytokine production razolium (NBT) test in the presence or absence of 1.67 by IFNg-differentiated U937 cells, but not in undifferentiated mm of 12-O- tetradecanoylphorbol-13-acetate (TPA) as U937. Interestingly, differentiation of these previously reported (Roberts et al., 1991). The percentage cells by IFNg appears to upregulate the expression of of cells containing formazan in each sample was deter- PAR1 and specific binding of thrombin to these cells. mined by counting at least 300 cells (undifferentiated Thus, thrombin may play an important role in regulating U937 cells, Ç1%; IFNg-differentiated U937 cells, Ç50%). differentiated monocytic functions in these cells at sites of tissue repair, inflammation, or even in atherosclerotic Northern blot analysis plaques. Total RNA was extracted from undifferentiated and IFNg-differentiated U937 cells using TRI REAGENT MATERIALS AND METHODS (Molecular Research Center, Inc., Cinncinnati, OH), Reagents separated on a 5.4% formaldehyde, 1% agarose gel, and Highly purified human a-thrombin (99% a form) was blotted onto nylon membrane (Micron Separations Inc., a kind gift from Dr. J.W. Fenton (Albany, NY) (specific Westboro, MA). The membrane was UV crosslinked. activity 3,683 NIH U/mg protein). Another preparation Human a-thrombin receptor probe (PAR1) (a kind gift of highly purified human a-thrombin (specific activity from Dr. M.S. Runge, Galveston, TX) or G3PDH obtained 4,000 NIH U/mg protein) and 3-(4,5- dimethylthiazol- from Clontech (Palo Alto, CA) was then radiola- 2yl)-2,5-diphenyl tetrazolium bromide (MTT) were pur- beled with 32 P-dCTP obtained from Amersham (Arlington chased from Sigma (St. Louis, MO). Recombinat human Heights, IL) using a Decaprime labeling kit (Amchased IFNg was obtained from Genentech (San Francisco, CA) bion Inc., Austin, TX). After hybridization in 50% with a specific activity of IU/mg protein. formamide, 51 SSPE, 0.1% SDS, 51 Denhardt s, and Cell separation 100 mg/ml sheared salmon sperm DNA at 55 C overnight (18 hr), the nylon membrane was washed under Peripheral blood from healthy donors was used as a high stringency conditions (0.11 SSC, 1% SDS) and source of mononuclear cells (PBMC) and was applied exposed to a phosphoimaging screen overnight (18 hr; directly to a gradient of Ficoll-Hypaque (Pharmacia, Molecular Dynamics, Sunnyvale, CA). Bands were Piscataway, NJ) as previously described (Naldini et al., quantitated using the Image Quant software available 1997). Monocyte-depleted PBMCs were obtained as follows. within the phosphoimager. PBMCs were passed over nylon wool columns and the eluted cells were further incubated once or twice Magnetic bead sorting on plastic in culture grade flasks (Costar, Cambridge, undifferentiated and IFNg-differentiated MA) at 37 C for 2 hr in RPMI (GIBCO, Grand Island, U937 cells were placed in serum-free media for 2 hr. NY) and 2% heat-inactivated fetal calf serum (FCS). Cells were then centrifuged for 9 min at 1,000 rpm Nonadherent cells were collected, washed three times and resuspended in Dulbecco modified Eagle mewith HYQ-CCM1 serum-free medium (Hyclone, Logan, dium:ham s F12 nutrient mixture (1:1) (DV) with 15 UT), and resuspended for culture ( /100 ml). The mm Hepes, 1% bovine serum albumin (BSA) at 1 1

3 78 NALDINI ET AL. TABLE 1. Effect of thrombin and FCS on undifferentiated U937 cells Medium Thrombin FCS { { { 4.9* 1 Results are expressed as O.D. % U937 cells were cultured in 100 ml of serum-free medium (HYQ-CMM1) with the addition of either thrombin (10 mg/ml) of FCS (0.25%) for 1 hr at 37 C. An additional 100 ml of medium supplemented with FCS (1%) was added. Following 72 hr, 100 ml of culture medium was removed from each well and 10 ml of MTT was added. After 4 hr incubation, 100 ml of acid propan-2-ol was added and the O.D. was assessed as described in Materials and Methods. Data represent a typical experiment performed in quadruplicate. *Statistically significant (P õ 0.05) difference between O.D.% of cultures treated with either thrombin or FCS vs. untreated cultures. Filters were counted in a gamma counter and specific binding was determined by subtracting the nonspecific binding from total counts. Proliferation assays The proliferative responses of U937 cells to thrombin were performed using the MTT colorimetric method (Mosmann, 1983). Briefly, U937 or IFNg-differentiated Fig. 1. Thrombin enhances IFNg-differentiated U937 proliferation. U937 cells (10 5 cells/well) were incubated in 100 ml se U937 and IFNg- differentiated U937 cells were cultured in 100 ml rum-free medium, in 96-well microtiter plates at 37 C of serum-free medium (HYQ-CMM1) without (open bars) or with 5 mg/ml of thrombin (filled bars) for 1 hr at 37 C. An additional 100 ml in the presence of varying concentrations of thrombin. of medium supplemented with FCS (1%) was added. Following 72 hr, One hour later, 100 ml of medium supplemented with 100 ml of culture medium was removed from each well and 10 ml of 2% FCS was added and incubation continued. Follow- MTT was added. After 4 hr incubation, 100 ml of acid propan-2-ol ing indicated time points, 100 ml of culture medium was added and the O.D. was assessed as described in Materials and Methods. Data represent the M { SEM of three independent experisolution. Four hours later, 100 ml of acid was removed and cells were treated with 10 ml ofmtt ments performed in quadruplicate. Asterisk indicates statistically sig- propan-2-ol nificant (P õ 0.05) differences between O.D.% of cultures treated with thrombin vs. untreated cultures. (0.04 M HCl in propan-2-ol) was added to dissolve the formazan product. The microplates were read using a microelisa reader at 570 nm using a reference wavelength of 630 nm and a calibration setting of /ml cells were aliquoted into 1.5 ml micro- Cytokine assays fuge tubes and incubated at 22 C for 10 min. 60 ng/ml biotinylated a-thrombin was added and the incubation IL6, IL1b, TNFa, and IL8 were assessed from cell- continued for 90 min at 22 C. Streptavidin-coated magassay (ELISA). Briefly, U937 or IFNg-differentiated free supernatants by enzyme-linked immunosorbent netic beads (CPG, Lincoln Park, NJ) were washed and resuspended in binding buffer (10 mm phosphate, ph U937 cells, PBMC, monocytes, and monocyte-depleted 7.5) at 0.56 mg/ml. Beads were added such that the PBMC were cultured with and without thrombin at bead:cell ratio was approximately 3:1 (0.225 mg) and the indicated concentrations in 100 ml of serum-free incubation was continued for 1 hr with rotation at 22 C. medium as described above. One hour later, 100 ml of Cells were magnetically separated and the supernatant medium containing 2% FCS was added and incubation was aspirated. Magnetic beads with or without cells continued. At the appropriate intervals, cell culture su- attached were washed two to three times by adding 1 pernatants were harvested and placed at -20 C pending ml of washing buffer (10 mm phosphate, ph 7.5, 1 M assay. ELISA kits were obtained from Biosource Inter- NaCl, 0.1% BSA) followed by magnetic separation. national (Camarillo, CA) and R&D Systems (Minneap- After the final wash, beads were resuspended in 1 ml of olis, MN) and did not show cross-reactivity with other washing buffer diluted in 9 ml PBS-azide and counted. cytokines (õ0.0005%). Minimum detectable doses were More than 99% of the cells were viable following this as follows: IL6 Å 2 pg/ml; IL1b Å 3 pg/ml; TNFa Å 4.4 assay as measured by trypan blue exclusion. pg/ml; IL8 Å 3 pg/ml. Coefficients of variation for intra- assay precision were õ5%. 125 I-thrombin binding Statistics (116 mg of protein) U937 or IFNg-differentiated U937 (110 mg of protein) were placed in serumfree medium for 2 hr and then resuspended in 0.1 ml DV-Hepes/1% BSA in 1.5 ml microfuge tubes and incubated for 10 min at 22 C. 125 I-thrombin (60 ng/ml) was added to each tube to determine total binding. Nonspe- cific binding was assessed by adding 100-fold excess of cold a-thrombin to 60 ng/ml I 125 -thrombin. Cells were incubated for 2 hr at 22 C and then filtered through Whatman GF/C filters followed by washing with PBS. Unless otherwise stated, results are expressed as percent of control. Data represent the mean { SEM. Statistical analysis was by Student s two-tailed t-test. RESULTS Effects of thrombin on differentiated and undifferentiated U937 cells To evaluate whether thrombin differentially affects the proliferation of monocyte-differentiated and undif- ferentiated cells, IFNg-treated and control U937 cells

4 THROMBIN-INDUCED MONOCYTE ACTIVATION 79 Fig. 2. A: Dose response of thrombin enhancement of IFNg-differen- B: Kinetics of thrombin-induced proliferation. IFNg-differentiated tiated U937 proliferation. IFNg-differentiated U937 cells were incu- U937 cells were incubated with HYQ-CCM1 medium or thrombin (10 bated with HYQ-CMM1 medium or varying concentrations of throm- mg/ml) for 1 hr at 37 C, then supplemented with FCS (1%). After 24, bin for 1 hr at 37 C. An additional 100 ml of medium supplemented 48, and 72 hr, 100 ml of culture medium was removed from each well with FCS (1%) was added. Following 72 hr, 100 ml of culture medium and 10 ml of MTT was added. After 4 hr incubation, 100 ml of acid was removed from each well and 10 ml of MTT was added. After 4 hr propan-2-ol was added and the O.D. was assessed as described under incubation, 100 ml of acid propan-2-ol was added and the O.D. was Materials and Methods. Data are the M { SEM of three independent assessed as described in Materials and Methods. Data are the M { experiments performed in quadruplicate. Asterisks indicate statisti- SEM of three independent experiments performed in quadruplicate. cally significant (P õ 0.05) differences between O.D.% of cultures Asterisks indicate statistically significant (P õ 0.05) differences be- treated with thrombin vs. untreated. tween O.D.% of cultures treated with thrombin vs. untreated cultures. were incubated in the presence or absence of thrombin IFNg-treated U937 cells are shown in Figure 2B. and proliferation was assessed using the MTT colorimetric Thrombin treatment induced significant proliferation assay. Treatment of IFNg-differentiated U937 of IFNg-differentiated U937 cells first observed at 24 hr cells with thrombin resulted in a significant increase which continued to increase to 72 hr of culture. These in proliferative response (Fig. 1). The increase in prolif- results show that thrombin stimulates proliferation of eration induced by thrombin was approximately 160% IFNg-differentiated U937 cells in a time and dose-de- greater than those cells treated with medium alone. It pendent manner, but that thrombin does not stimulate should be noted that in these experiments, there was proliferation in undifferentiated U937 cells. Moreover, a small increase in the proliferative response of undif- the effect of thrombin of differentiated U937 cells still ferentiated U937, even in the presence of 10 mg/ml persists when IFNg is left in the culture (Table 2). thrombin; however FCS was able to increase significantly the cell proliferation as shown in Table 1. Thrombin enhances IL6 production Thrombin induction of proliferation was concentration by U937 cells dependent with optimal proliferation observed using To further investigate thrombin enhancement of 5 10 mg/ml of thrombin (Fig. 2A). This concentration monocyte activation, we investigated whether thromof thrombin has also been shown to be necessary for T- bin treatment of IFNg-differentiated U937 cells resulted lymphocyte and monocyte thrombin-induced stimulatory in enhanced IL6 production. IFNg-differentiated effects (Clohisy et al., 1990; Naldini et al., 1993). U937 were treated with or without thrombin and assessed The kinetics of the thrombin-induced proliferation of for IL6 production. Thrombin induced IL6 pro-

5 80 NALDINI ET AL. TABLE 2. Effect of thrombin on differentiated U937 cells in the presence of IFNg Thrombin (mg/ml) Medium TABLE 3. Thrombin enhances IL6 production in human monocytes 1 Culture conditions IL6 production Thrombin (mg/ml) IL6 (pg/ml) % of control { { { 2.5* 0 1,512 { { 3 1 Results are expressed as O.D.% differentiated U937 cells were cultured in 1 1,702 { { ml of serum-free medium (HYQ-CMM1) with addition of thrombin, in the 5 1,827 { 62* 143 { 17* presence of IFNg (100 IU/ml) for 1 hr at 37 C. An additional 100 ml of medium supplemented with FCS (1%) was added. Following 72 hr, 100 ml of culture 10 2,124 { 109* 167 { 22* medium was removed from each well and 10 ml of MTT was added. After 4 hr incubation, 100 ml of acid propan-2-ol was added and the O.D. was assessed 1 Human monocytes were incubated with serum-free medium (HYQ-CCM1) with as described in Materials and Methods. Data represent a typical experiment or without varying concentrations of thrombin for 1 hr at 37 C, then supple- performed in quadruplicate. mented with the same medium / FCS (1%). After 48 hr, cell-free supernatants *Statistically significant (P õ 0.05) difference between O.D.% of cultures treated were obtained and pooled from triplicate cultures. Results are expressed as with thrombin vs. untreated cultures. pg/ml of IL6 and as percent of control. Data presented are the M { SEM of four independent experiments. *Statistically significant (P õ 0.05) differences between IL6 produced in cultures treated with thrombin vs. untreated parallel cultures. duction in a dose-dependent manner (Fig. 3A). Although thrombin was ineffective at a concentration of 1 mg/ml, 10 mg/ml of thrombin induced a twofold inman lating the production of inflammatory cytokines by hucrease in the IL6 production by IFNg-differentiated monocytes. U937 cells. These data are in agreement with our proliferation experiments (Figs. 1, 2) as well as with previous Correlation between increased response to studies using T lymphocytes and PBMC (Naldini et al., thrombin and thrombin receptor expression 1993). The kinetics of IL6 production are presented in A number of receptors appear to be expressed on Figure 3B. Thrombin-induced IL6 production was detected various cells to which thrombin may bind to initiate as early as 24 hr posttreatment (80% increase) proliferative and cytokine releasing signals. We there- and increased slightly at 48 hr of culture (100% increase) fore set out to determine if IFNg- induced differentia- with a decline at 72 hr (60% increase). In these tion caused upregulation of either PAR1 or the specific experiments, thrombin treatment of undifferentiated binding of thrombin to cells. Monocytes have been U937 did not result in an increase of IL6 production shown to express PAR1 (Hoffman and Church, 1993). (data not shown). To determine whether PAR1 is expressed and upregulated during monocyte differentiation, U937 cells were Thrombin enhances cytokine production treated with and without IFNg for 10 days. Total RNA by peripheral blood monocytes was purified and subjected to Northern blot analysis Since thrombin induced IL6 production in IFNg-differentiated as described in the Materials and Methods section. U937 cells, we next investigated thrombin s U937 cells differentiated into monocytes expressed effect on cytokine production in human peripheral three to fivefold more PAR1 message than undifferenti- blood monocytes. Monocytes were treated with throm- ated U937 cells (Fig. 5). This increase is not seen in bin and cell-free supernatants from these cultures were G3PDH. Although cells treated with IFNg express a assessed for IL6, IL1b, TNFa, and IL8 production. IL6 spliced G3PDH message, quantitation shows that the production was dose dependent following thrombin hybridization to the PAR1 message is two to threefold treatment of monocytes (Table 3). These results are higher than that for either of the G3PDH bands. Thus, consistent with the results obtained in IFNg-differentiated these results indicate that upon differentiation, PAR1 U937 (Fig. 3). The increase in IL6 production in- expression is increased. duced by thrombin (10 mg/ml) was more than 60% Because there is evidence for moderate/high-affinity greater than control cultures without thrombin treat- receptor binding for thrombin on many cells (Carney ment. To further confirm the role of monocytes in the et al., 1992b), including those that do not express PAR1, thrombin-induced IL6 production, we performed exper- we wanted to determine whether specific thrombin iments using unfractionated PBMC and monocyte-depleted binding also increases with monocyte differentiation. PBMC (Table 4). Thrombin induced a threefold We have recently developed a nonradioactive assay increase in IL6 production by unfractionated PBMC that measures retention of thrombin on the surface of (more than a 120% increase) when compared with cells. The results of our nonradioactive assay are shown thrombin-treated monocyte-depleted PBMC which produced in Figure 6A. Undifferentiated and IFNg-differentiated no IL6. It should be noted that in these experi- U937 cells were incubated with or without biotinylated ments monocyte-depleted PBMC contain Ç90% T lymphocytes thrombin. Those cells that bound biotinylated thrombin (CD3/), 10% NK cells (CD 16/), and less than were separated from those that did not bind thrombin 0.1% monocytes (CD14/). Thrombin induction of cytokine by using streptavidin-coated magnetic beads. As shown production by monocytes was not limited to IL6. in Figure 6A, IFNg-differentiated cells had a threefold Monocytes treated with thrombin produced twofold increase in the number of cells binding thrombin when more IL1b and TNFa when compared to cultures compared with the number of undifferentiated cells treated with medium alone (Fig. 4). Interestingly, in binding thrombin. To determine whether the binding monocytes just as in IFNg-differentiated U937 (data of thrombin to differentiated U937 cells was a specific not shown), thrombin did not induce IL8 production. interaction, we initiated 125 I-thrombin binding studies. Thus, thrombin- induced cytokine production in human Figure 6B shows the increase in specific binding of 125 I- monocytes is comparable to that seen in U937 cells. thrombin to IFNg-differentiated vs. undifferentiated These results suggest a direct role for thrombin in regu- U937 cells. As shown with this assay as well, there is a

6 THROMBIN-INDUCED MONOCYTE ACTIVATION 81 Fig. 3. A: Dose response of thrombin enhancement of IFNg-differen- Kinetics of thrombin-induced IL6 production. IFNg-differentiated tiated U937 IL6 production. IFNg-differentiated U937 cells were incu- U937 cells were incubated with HYQ-CMM1 medium or varying conbated with HYQ-CMM1 medium or varying concentrations of throm- centrations of thrombin for 1 hr at 37 C, then supplemented with FCS bin for 1 hr at 37 C, then supplemented with FCS (1%). After 48 (1%). After 24, 48, and 72 hr, cell-free supernatants were obtained hr, cell-free supernatants were obtained and pooled from triplicate and pooled from triplicate cultures. Data presented are expressed as cultures. Data presented are expressed as % of control and are the % of control and are the M { SEM of three independent experiments. M { SEM of three independent experiments. Asterisks indicate statis- Asterisks indicate statistically significant (P õ 0.05) differences betically significant (P õ 0.05) differences between IL6 produced in tween IL6 produced in cultures treated with thrombin vs. untreated cultures treated with thrombin vs. untreated parallel cultures. B: cultures. TABLE 4. Effects of thrombin on IL6 production in PBMC 1 IL6 production Unfractionated PBMC Monocyte-depleted PBMC Culture conditions IL6 (pg/ml) % of control IL6 (pg/ml) % of control Medium 1,022 { { 21 õ2 n.a. 2 Thrombin 3,210 { 1, { 44* õ2 n.a. 1 Unfractionated and monocyte-depleted PBMC from the same buffy coat were cultured with serum-free medium (HYQ-CMM1) with and without 10 mg/ml of thrombin for 1 hr at 37 C, then supplemented with the same medium / FCS (1%). After 48 hr, cell-free supernatants were obtained and pooled from triplicate cultures. Results are expressed as pg/ml of IL6 and as percent of control. Data presented are the M { SEM of three independent experiments. 2 Not applicable. *Statistically significant (P õ 0.05) differences between IL6 produced in cultures treated with thrombin vs. untreated parallel cultures. threefold increase in specific thrombin binding to IFNgdifferentiated U937 cells. Thus, in both nonradioactive thrombin binding assays and assays with 125 I-thrombin, the thrombin binding to U937 cells is shown to increase by approximately threefold when these cells are differentiated toward monocytes. DISCUSSION Thrombin has been shown to mediate a variety of biological effects (Shuman, 1986). Many of the func- tions mediated by thrombin have also been shown to play important roles in wound healing and in inflam-

7 82 NALDINI ET AL. Fig. 5. Upregulation of PAR1 expression in IFNg-differentiated U937 cells. Total RNA from U937 (lane A) and IFNg-differentiated U937 cells (lane B) were treated as described in Materials and Methods. Quantitation was achieved by using the Image Quant program of the phosphoimager. Percent area for the top blot for lane A: 5; lane B: 38. Percent area for the bottom blot for lane A: 25; lane B, upper band: 15; lower band: 18. can be produced by a variety of cells and acts on a variety of tissues, exerting growth-inducing, growthinhibitory, and differentiation-inducing effects, depending on the target cell (Van Snick, 1990). In our studies, thrombin did not induce IL6 production by un- Fig. 4. Effects of thrombin on cytokine production by human mono- differentiated U937 or monocyte-depleted PBMC, but cytes. Human monocytes were incubated with HYQ-CMM1 medium did induce IL6 by IFNg-differentiated U937 cells. These (open bars) or 5 mg/ml of thrombin (filled bars) for 1 hr at 37 C, then supplemented with FCS (1%). After 48 hr, cell-free supernatants were results were unexpected as it has been shown that obtained and pooled from triplicate cultures. Data presented are expressed monocytes do not produce IL6 upon thrombin stimula- as % of control and are the M { SEM of three independent tion (Sower et al., 1996); however, the time points when experiments. Asterisks indicate statistically significant (P õ 0.05) supernatants were collected differ between the two differences between cytokines produced in cultures treated with thrombin vs. untreated cultures. studies. In these experiments, supernatants were col- lected at 48 hr, whereas in the previous studies, supernatants were collected at 8 hr. While the production of IL6 is apparent, these results suggest that the cytokine mation (Carney et al., 1992a). In this report, we now production is cell cycle dependent or may be a result show that with differentiation of U937 cells toward of cell proliferation or a secondary event rather than monocytes, cells become competent to respond to direct thrombin stimulation. Regardless, these results thrombin with cytokine production and enhanced cell suggest that monocytes are the cells involved in the proliferation. With this differentiation, thrombin binding production of IL6 by PBMC. Unexpectedly, thrombin and PAR1 expression both increase by approxi- did not induce IL8 production by monocytes under the mately threefold. Thus, as monocytes differentiate, it conditions used for these studies. It is possible that appears that they become more responsive to thrombin. the IL8 production induced by thrombin at early time Clohisy et al. (1990) have shown that thrombin was points (Sower et al., 1996) is turned off by the cells by not mitogenic for normal murine bone marrow-derived 48 hr or that the IL8 has been degraded. These data macrophages. However, thrombin did greatly enhance make sense physiologically because in early wounding macrophage proliferation in response to CSF-1. These the IL8 production induced by thrombin may be necessary results are similar to our findings using human to recruit other cells into the wound, but after 48 PBMCs, monocytes, and U937 cells. Thrombin was hr this recruitment may no longer be needed. These rarely mitogenic to undifferentiated U937 cells but did data further implicate thrombin as an important medi- greatly enhance proliferation of IFNg-differentiated ator of the inflammatory response. U937. In agreement with our findings, Bar-Shavit et The mechanism by which thrombin induces enal. (1987) reported that thrombin was chemotactic for hanced proliferation is not yet understood. High/moderate the human leukemic cell line HL60 which was induced affinity thrombin receptors, such as those originally to differentiate along the monocytic lineage. reported on fibroblasts (Rasmussen et al., 1991), have Thrombin is capable of inducing various cells to produce been identified in a number of cell types (Carney et al., a number of proinflammatory cytokines which me- 1992b) including macrophages (Kudahl et al., 1991), diate induction of different biological responses. Endothelial neutrophils (Bizios et al., 1986), T lymphocytes (Tordai cells stimulated with thrombin synthesize plate- et al., 1993), and NK cells (Howells et al., 1993). There let-derived growth factor (PDGF; Harlan et al., 1986), is evidence that this high-affinity binding does not cor- IL1 (Stern et al., 1985), prostaglandins (Weksler et al., relate with expression of PAR1, suggesting that highaffinity 1978), and plasminogen activators (Levin et al., 1984). binding may be to a separate N-PAR compo- Thrombin was also shown to greatly enhance IL1 pro- nent. The present study confirms that human monocytes duction by lipopolysaccharide (LPS)-stimulated macrophages and differentiated U937 cells bind thrombin and (Jones and Geczy, 1990). We recently reported express PAR1. Previous studies have shown that func- that thrombin can activate human T lymphocytes and enhance cytokine (IL6 and IL2) production by PBMC (Naldini et al., 1993). IL6 is a pleiotropic cytokine that tional thrombin receptors are present on U937 cells (Joseph and MacDermot, 1993), but there had been no evidence presented that these cells bind thrombin with

8 THROMBIN-INDUCED MONOCYTE ACTIVATION 83 Fig. 6. A: Increase in specific thrombin binding upon U937 differenti- using duplicate samples. B: U937 and IFNg-differentiated U937 cells ation. U937 and IFNg-differentiated U937 cells were incubated with were incubated with 125 I-thrombin for 90 min { 100-fold excess cold biotinylated thrombin (hatched bars) or without biotinylated throm- thrombin and filtered through GF/C filters as described in Materials bin (unhatched bars) for 90 min as described in Materials and Meth- and Methods. Specific binding Å total counts - nonspecific counts. ods. Thrombin binding was determined by cell sorting using streptavi- Asterisk indicates statistical significance where P õ Data are din-coated magnetic beads. Asterisk indicates statistical significance representative of three independent experiments using duplicate sam- (P õ 0.05). Data are representative of two independent experiments ples. high enough affinity to measure in or 125 I-thrombin duction (Naldini et al., 1993). We now show that differentiated binding or nonradioactive thrombin binding experiments. monocytes are activated by thrombin to probinding Consistent with these findings, in HL60 cells, duce IL6, IL1b, and TNFa. In addition, thrombin monocytic-differentiated lineage appears to correlate induces IL6 production in fibroblasts and IL6 enhances with enhanced high-affinity thrombin binding (Bar- the thrombin receptor expression in these cells (Sower Shavit et al., 1987). Additional experiments are now et al., 1995). Thus, the ability of thrombin to induce underway to establish if the increased thrombin bind- the production of fibrogenic cytokines (IL6, IL1b, and ing observed in our studies could be due to the increased TNFa) by monocytes could have important physiologiin expression of PAR1 or if it is due to the increase cal consequences in the orchestration of wound healing expression of the N-PAR component. and the inflammatory response than previously recog- Physiologically, thrombin enhancement of monocyte nized. activation could be extremely important in regulating the wound healing process and subsequent events triggered ACKNOWLEDGMENTS by release of IL6 and other cytokines (Sporn and The authors thank Dr. F. Carraro and Prof. G.P. Pes- Roberts, 1993; Slavin, 1996). Indeed, IL6, IL1b, and sina for valuable discussion and Mrs. P. Marrocchesi TNFa are known as fibrogenic cytokines (Kovacs, 1991) for assistance with the manuscript preparation. because they promote later wound healing responses. Thrombin is always present in the wound. Following LITERATURE CITED clot formation, wound healing proceeds involving a se- Bar-Shavit, R., Kahn, A., Fenton, J.W.I., and Wilner, G.D. (1983) ries of events. First, neutrophils, lymphocytes, and Chemotactic response of monocytes to thrombin. J.Cell Biol., monocytes accumulate into the area of the wound. 96: Next, fibroblasts and endothelial and epithelial cells Bar-Shavit, R., Kahn, A.J., Mann, K.G., and Wilner, G.D. (1986) Identification of a thrombin sequence with growth factor activity on migrate into the wound, where they proliferate. We macrophages. Proc. Natl. Acad. Sci.USA, 83: have previously shown that thrombin activates T lym- Bar-Shavit, R., Hruska, K.A., Kahn, A.J., and Wilner, G.D. (1987) phocytes, enhancing T-cell proliferation and IL6 pro- Thrombin chemotactic stimulation of HL-60 cells: Studies on throm-

9 84 NALDINI ET AL. bin responsiveness as a function of differentiation. J.Cell. Physiol., bin stimulates tissue plasminogen activator release from cultured 131: human endothelial cells. J.Clin. Invest., 74: Bizios, R., Lai, L., Fenton, J.W.II., and Malik, A.B. (1986) Thrombin- Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and induced chemotaxis and aggregation of neutrophils. J.Cell. Physiol., survival: Application to proliferation and cytotoxicity assays. J. Im- 128: munol. Method., 65: Brass, L.F., Manning, D.R., Williams, A.G., Woolkalis, M.J., and Naldini, A., and Carney, D.H. (1996) Thrombin modulation of natural Poncz, M. (1991) Receptor and G protein-mediated responses to killer activity in human peripheral lymphocytes. Cell. Immunol., thrombin in HEL cells. J.Biol.Chem., 266: : Carney, D.H., and Cunningham, D.D. (1978) Cell surface action of Naldini, A., Carney, D.H., Bocci, V., Klimpel, K.D., Asuncion, M., thrombin is sufficient to initiate division of chick cells. Cell, 14:811 Soares, L.E., and Klimpel, G.R. (1993) Thrombin enhances T cell 823. proliferative responses and cytokine production. Cell. Immunol., Carney, D.H., Herbosa, G.J., Stiernberg, J., Bergmann, J.S., Gordon, 147: E.A., Scott, D., and Fenton, J.W.I. (1986) Double-signal hypothesis Naldini, A., Carraro, F., Silvestri, S., and Bocci, V. (1997) Hypoxia for thrombin initiation of cell proliferation. Semin.Thromb. Hemost., affects cytokine production and proliferative responses by human 12: peripheral mononuclear cells. J.Cell. Physiol., 173: Carney, D.H., Mann, R., Redin, W.R., Pernia, S.D., Berry, D., Heggers, Oberg, F., Larsson, L.-G., Anton, R., and Nilsson, K. (1991) Interferon J.P., Hayward, P.G., Robson, M.C., Christie, J., Annable, C., Fenton gamma abrogates the differentiation block in v-myc-expressing U- II, J.W., and Glenn, K.C. (1992a) Enhancement of incisional wound 937 monoblasts. Proc. Natl. Acad. Sci.USA, 88: healing and neovascularization in normal rats by thrombin and Rasmussen, U.B., Craviari, V.V., Jallat, S., Schlesinger, Y., Pages, G., synthetic thrombin receptor-activating peptides. J.Clin. Invest., Pavirani, A., Lecocq, J.-P., Pouyssegur, J., and Van Obberghen- 89: Shilling, E. (1991) DNA cloning and expression of a hamster a- Carney, D.H., Redin, W., and McCroskey, L. (1992b) Role of highaffinity thrombin receptor coupled to Ca 2/ mobilization. FEBS Lett., thrombin receptors in postclotting cellular effects of throm- 288: bin. Semin.Thromb. Hemost., 18: Roberts, P.J., Devalia, V., Faint, R., Pizzey, A., Bainton, A.L., Thomas, Clohisy, D.R., Erdmann, J.M., and Wilner, G.D. (1990) Thrombin N.S.B., Pilkington, G.R., and Linch, D.C. (1991) Differentiationlinked binds to murine bone marrow-derived macrophages and enhances activation of the respiratory burst in a monocytic cell line colony-stimulating factor-1-driven mitogenesis. J. Biol. Chem., (U937) via Fc trii. A study of activation pathways and their regulation. 265: J.Immunol., 147: Colotta, F., Sciacca, F.L., Sironi, M., Luini, W., Rabiet, M.J., and Shuman, M.A. (1986) Thrombin-cellular interactions. Ann. N.Y. Acad. Mantovani, A. (1994) Expression of monocyte chemotactic protein- Sci., 485: by monocytes and endothelial cells exposed to thrombin. Am. J. Slavin, J. (1996) The role of cytokines in wound healing. J. Pathol., Pathol., 144: :5 10. Gardner, I.D., and Remington, J.S. (1978) Aging and the immune Sower, L.E., Froelich, C.J., Carney, D.H., Fenton, J.W., and Klimpel, response. II. Lymphocyte responsiveness and macrophage activaepithelial G.R. (1995) Thrombin induces IL-6 production in fibroblasts and tion in Toxoplasma gondii-infected mice. J. Immunol., 120: cells - Evidence for the involvement of the seven-trans- Gospodarowicz, D., Brown, K.D., Birdwell, C.R., and Zetter, B.R. membrane domain (STD) receptor for alpha-thrombin. J. Immunol, (1978) Control of proliferation of human vascular endothelial cells. 155: Characterization of the response of human umbilical vein endotheand Sower, L.E., Froelich, C.J., Allegretto, N., Rose, P.M., Hanna, W.D., Klimpel, G.R. (1996) Extracellular activities of human gran- lial cells to fibroblast growth factor, epidermal growth factor, and thrombin. J.Cell Biol., 77: zyme A - Monocyte activation by granzyme A versus alpha-throm- Grand, R.J.A., Turnell, A.S., and Grabham, P.W. (1996) Cellular con- bin. J. Immunol., 156: sequences of thrombin-receptor activation. Biochem.J., 313:353 Sporn, M.B., and Roberts, A.B. (1993) A major advance in the use of 368. growth factors to enhance wound healing. J.Clin. Invest., 92 :2565 Gurwitz, D., and Cunningham, D.D. (1988) Thrombin modulates and reverses neuroblastoma neurite outgrowth. Proc. Natl.Acad. Sci. Stern, D.M., Bank, I., Nawroth, P.P., Cassimeris, J., Kisiel, W., Fen- USA, 85: ton, J.W.I., Dinarello, C., Chess, L., and Jaffe, E.A. (1985) Self- Harlan, J.M., Thompson, P.J., Ross, R.R., and Bowen-Pope, D.F. regulation of procoagulant events on the endothelial cell surface. J. (1986) Alpha thrombin induces release of platelet-derived growth Exp. Med., 162: factor-like molecule(s) by cultured human endothelial cells. J.Cell Sundström, C., and Nilsson, K. (1976) Establishment and character- ization of a human histiocytic lymphoma cell line. Int. J. Cancer, Biol., 103: : Hoffman, M., and Church, F.C. (1993) Response of blood leukocytes Tordai, A., Fenton, J.W.I., Andersen, T., and Gelfand, E.W. (1993) to thrombin receptor peptides. J. Leukoc. Biol., 54: Functional thrombin receptors on human T lymphoblastoid cells. Howells, G.L., Macey, M., Curtis, M.A., and Stone, S.R. (1993) Periph- J. Immunol., 150: eral blood lymphocytes express the platelet-type thrombin receptor. Ucla, C., Roux-Lombard, P., Fey, S., Dayer, J.-M., and Mach, B. (1990) Br. J. Haematol., 84: Interferon gamma drastically modifies the regulation of interleukin Jenkins, A.L., Howells, G.L., Scott, E., Le Bonniec, B.F., Curtis, M.A., 1 genes by endotoxin in U937 cells. J. Clin. Invest., 85: and Stone, S.R. (1995) The response to thrombin of human neutro- Van Snick, J. (1990) Interleukin-6: An overview. Annu. Rev. Immuphils: Evidence for two novel receptors. J.Cell Sci., 108: nol., 8: Jones, A., and Geczy, C.L. (1990) Thrombin and factor Xa enhance Vouret-Craviari, V., Van Obberghen Schilling, E., Rasmussen, U.B., the production of interleukin-1. Immunology, 71: Pavirani, A., Lecocq, J.-P., and Pouyssegur, J. (1992) Synthetic Joseph, S., and MacDermot, J. (1993) The N-terminal thrombin recep- alpha-thrombin receptor peptides activate G protein-coupled signaltor fragment SFLLRN, but not catalytically inactive thrombin-de- ing pathways but are unable to induce mitogenesis. Mol. Biol.Cell, rived agonists, activate U937 human monocytic cells: Evidence for 3: receptor hydrolysis in thrombin-dependent signalling. Biochem. J., Weiss, R.H., and Nuccitelli, R. (1992) Inhibition of tyrosine phosphory- 290: lation prevents thrombin-induced mitogenesis, but not intracellular Kovacs, E.J. (1991) Fibrogenic cytokines: The role of immune media- free calcium release, in vascular smooth muscle cells. J. Biol. Chem., tors in the development of scar tissue. Immunol.Today, 12: : Kudahl, K., Fisker, S., and Sonne, O. (1991) A thrombin receptor in Weksler, B., Ley, C.W., and Jaffe, E.A. (1978) Stimulation of endotheresident rat peritoneal macrophages. 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