Heparanase powers a chronic inflammatory circuit that promotes colitis-associated tumorigenesis in mice (original) (raw)
Overexpression of heparanase in clinical and experimental chronic colitis
Applying immunohistochemical staining to examine heparanase expression in colon specimens from UC patients (n = 10), as well as in control samples derived from healthy colonic tissue specimens (n = 29) (Figure 1), we extended our previous report (16) on preferential expression of heparanase in UC. Notably, while heparanase expression was barely detected in healthy colon epithelium (negative staining in 24 biopsies and faint staining in 5 biopsies) (Figure 1A), in UC specimens the epithelial lining showed a marked expression of heparanase in both the acute and chronic phases of the disease (strong staining in all biopsies; Figure 1A). Given the suggested role of heparanase in colon tumorigenesis (12–14, 23), it is important to note that a strong heparanase staining was detected during the chronic phase in the immature glands that had recently formed in the healing mucosa and that displayed initial dysplastic-like changes typical for this phase of UC (Figure 1A). In all phases, inflammatory cells in the involved areas showed little or no heparanase expression. We next studied the tempo-spatial pattern of heparanase expression in DSS-induced colitis. Repeated oral administration of DSS-supplemented water to mice is a reliable model of UC, recapitulating characteristic changes observed in the colon of UC patients. DSS causes an acute inflammatory reaction and ulceration in the colon (24), and when 3 cycles (7 days each, with 14-day intervals) of DSS administration are applied, acute inflammation is followed by chronic colitis (24). Moreover, 3 cycles of DSS subsequent to a single AOM pretreatment result in development of colon tumors in nearly 100% of the treated mice (19), representing a well-established model of colitis-associated cancer. In addition, DSS alone may cause colonic tumors in almost 50% of the treated mice when the DSS cycles are repeatedly administered 9 times (25), further demonstrating the relevance of this model to inflammation-induced tumorigenesis. In our initial experiments BALB/c mice were given 3 cycles of DSS-supplemented water, as indicated in Figure 1B. Mice were sacrificed on experimental day 110 and their colon excised, and heparanase expression was assessed by real-time PCR, Western blotting, and immunostaining. A marked increase in heparanase mRNA (Figure 1C), protein (Figure 1D, and Figure 1, E and G), and enzymatic activity (Figure 1F) was readily detected in the colon during all phases of DSS-induced colitis. Moreover, Western blot analysis revealed an even higher increase in the amount of proteolytically activated 50-kDa heparanase at all phases of DSS colitis (Figure 1, D and E). The 50-kDa enzyme represents an N-terminally processed heparanase, which is at least 100-fold more active than the 65-kDa proenzyme (7). CatL is the predominant protease responsible for processing and activation of proheparanase (10). In accordance, increased levels of CatL mRNA (Figure 1C) and protein (Figure 1, D and G) were detected in the mouse colonic tissue at corresponding time points (discussed below).
Expression of heparanase and CatL is induced during the course of colitis. (A) Heparanase expression in acute and chronic phases of UC. Tissue specimens derived from normal colon tissue (left) and UC patients in acute (middle) and chronic (right) phases of the disease were stained with anti-heparanase antibody (red staining). Photographs are representative of control (n = 29) and UC (n = 10) samples (original magnification, ×200). (B) Schematic representation of mouse model of DSS colitis (top) and AOM/DSS-induced colitis–associated carcinoma (bottom), induced as described in Methods. Mouse colonic tissues were harvested at indicated time points and analyzed as described below for C–G. (C) Heparanase (Hpa; left) and CatL (right) mRNA expression during the course of DSS-induced colitis, measured by qRT-PCR and normalized to actin mRNA (n = 5) *P < 0.05, **P < 0.01, ***P < 0.001. (D) Heparanase and CatL protein levels. Lysates of distal colons were harvested at the indicated time points and analyzed for heparanase and CatL protein by immunoblotting. Pro-, latent 65 kDa proenzyme; Active-, enymatically-active 50 kDa heparanase. (E) Densitometric quantification of total (65 kDa and 50 kDa, left) and active (50 kDa, right) heparanase presented in D. (F) Heparanase activity. Punch biopsies were harvested on day 59 from DSS-untreated (Cont) and treated (DSS) mice and cultured for 24 hours. Heparanase activity was determined in the conditioned medium. (G) Immunostaining (brown) of mouse colonic serial tissue sections with anti-heparanase (top) and CatL (bottom) antibodies. Note heparanase staining in the epithelial compartment and CatL staining in the stromal compartment (original magnification, ×200).
Heparanase overexpression increases the incidence and severity of colitis-associated tumors
We next applied the AOM/DSS model to investigate the possible contribution of heparanase to colitis-associated carcinoma. Unlike UC patients, in which increased heparanase expression is constantly preserved (Figure 1A), in DSS colitis, heparanase levels gradually decreased during the chronic phase (Figure 1, C–E). Thus, to assess the precise role of heparanase in colitis-related tumorigenesis, we utilized heparanase transgenic (Hpa Tg) mice along with their WT counterparts. Hpa Tg mice (26) have been extensively used in studies on the role of heparanase in angiogenesis (27), bone turnover (28), diabetic nephropathy (29), arterial stenosis (30), and amyloid diseases (31). Hpa Tg mice express elevated levels of heparanase in colonic epithelium (Supplemental Figure 1, A–D; supplemental material available online with this article; doi:10.1172/JCI43792DS1), but not in splenocytes (26), which closely resembles the heparanase expression pattern in UC patients. Of note, analysis of colonic tissue sections immunostained with antibodies directed against proliferating cell nuclear antigen (PCNA) and activated (cleaved) caspase-3 (Supplemental Figure 1E) revealed no differences in proliferation/apoptosis rate in colonic epithelium of healthy Hpa Tg versus WT mice.
A single dose of AOM was injected to 10-week-old Hpa Tg and WT mice prior to 3 cycles of DSS treatment (as indicated in Figure 1B and described in Methods). Mice were sacrificed on day 110 of the experiment and their colon removed and examined for tumor formation. Easily detectable colonic tumors, graded by histological examination as adenocarcinomas (well- to moderately differentiated), were present in all colons (Figure 2, A and B). However, a macroscopically apparent and statistically significant increase in tumor incidence (P = 0.026) was readily detected in Hpa Tg mice as compared with their WT littermates (Figure 2, A–C). In addition, heparanase overexpression markedly enhanced the severity of colitis-associated colonic tumors, as evidenced by increased tumor size (P = 0.020; Figure 2C), vascular density (Figure 2, E and F), and considerable augmentation in the shift of β-catenin from the cell membrane to a cytoplasmic/nuclear localization, observed in Hpa Tg mice compared with WT mice (Figure 2D). Moreover, when 3 DSS cycles were administered alone, without AOM pretreatment, none of the WT mice developed colonic tumors during the observation period (although pre-cancerous changes were noted in 25% of WT mice), whereas colonic tumors (mostly moderately differentiated adenocarcinomas) were easily seen by histological examination in 90% of the Hpa Tg mice (Figure 2, G and H). Notably, mice treated with AOM alone (without DSS) did not develop tumors during the observation period regardless of genotype, emphasizing the essential role of inflammation in this experimental system.
Overexpression of heparanase confers increased susceptibility to colitis-associated tumors in AOM/DSS-treated and DSS-treated mice. (A–F) AOM/DSS-treated mice. (G and H) DSS-treated mice. (A) Gross appearance. Colons (n = 9) were removed on day 110 from AOM/DSS-treated mice, opened longitudinally, washed in PBS, and photographed. (B) Sub-gross examination (original magnification, ×3) and (C) quantification of colonic tumors detected on day 110 (AOM/DSS-treated mice) revealed increased tumor incidence (C, top) and burden (C, bottom) in Hpa Tg colon (gray bars) versus WT colon (black bars) (n = 9). (D) Immunostaining with anti–β-catenin antibody reveals enhanced expression and nuclear localization of β-catenin in Hpa Tg (bottom) versus WT (top) colonic tumors (original magnification, ×400). (E and F) Increased vascular density in Hpa Tg versus WT colonic tumors evaluated by immunostaining with anti-CD31 antibody (original magnification, ×400) (E) and quantified by blind counting of blood vessels per microscopic field (F). (G and H) DSS-induced chronic colitis (without AOM administration) results in colonic tumor formation in Hpa Tg but not in WT mice. (G) Representative microphotographs of histological sections showing the presence of tumors in colons derived from Hpa Tg (bottom) but not WT (top) mice. Magnification, ×100; inset: boxed regions shown at higher magnification (×400). (H) Number of tumors per colon (top) and percentage of tumor-bearing animals (bottom) in Hpa Tg versus WT mice. Error bars represent mean ± SD. *P < 0.05, **P < 0.01.
Heparanase overexpression preserves chronic inflammatory conditions in DSS colitis
We investigated whether the increased tumor incidence and severity in Hpa Tg mice is due to an exacerbated colonic inflammatory phenotype. Interestingly, during the acute phase of DSS colitis (days 1–10 of the experiment; ref. 24), we were unable to detect a more severe inflammatory phenotype in Hpa Tg compared with WT mice (Supplemental Figure 2). The rate of weight loss (a characteristic feature of acute DSS colitis; ref. 24) was not statistically significant different between Hpa Tg and WT mice (Supplemental Figure 2A). Moreover, in both Hpa Tg and WT mice a similar degree of leukocyte infiltration and epithelial cell damage (including multifocal dropouts of entire crypts and ulceration) were seen in all parts of the colon (Supplemental Figure 2B). Immunostaining revealed no differences in the extent of infiltration of neutrophils, the first recruited effectors of the acute inflammatory response (32, 33), between Hpa Tg and WT mice on day 10 of the experiment (Supplemental Figure 2B). In both genotypes the same degree of increase in mRNA encoding for pro-inflammatory proteins (i.e., TNF-α, iNOS, COX-2) was observed during the acute phase of DSS inflammation (Supplemental Figure 2C). ELISA determination of TNF-α secreted ex vivo by cultured colon punch biopsies revealed 13.8 ± 7.3 pg/ml in Hpa Tg compared with 33.4 ± 3.5 pg/ml in WT mice, further demonstrating the lack of exacerbated acute inflammation in Hpa Tg mice.
In contrast to the acute phase, heparanase overexpression profoundly affected the chronic phase of DSS-induced colitis, as demonstrated by the different inflammatory phenotypes expressed by Hpa Tg and WT mice on experimental day 80 (1 month after cessation of the last DSS cycle). Microscopic examination of H&E-stained colonic sections revealed persistent mucosal infiltration by immunocytes in Hpa Tg mice compared with little or no infiltration in WT mice (Figure 3B). This infiltration was accompanied by high occurrence of dysplastic glands (Figure 3B). Since angiogenesis is recognized as an integral component of chronic inflammation in both human UC (34) and experimental mouse colitis (35), we assessed the vascular density, applying immunostaining with antibodies directed against CD31 (Figure 3C), and found that colons of Hpa Tg mice contained significantly more blood vessels compared with WT mice (Figure 3J). There was no difference in vascular density between colonic tissues derived from healthy untreated WT and Hpa Tg mice (data not shown).
Heparanase preserves chronic inflammatory conditions in DSS colitis. Colons were obtained from DSS-treated WT (left) and Hpa Tg (right) mice on day 80 and processed for immunohistochemistry (original magnification, ×200) with anti-heparanase antibody (A); H&E staining (B); immunohistochemistry with antibodies directed against CD31 (C), pIκBα (D), COX-2 (E), and cyclin D1 (G); and immunofluorescence with antibodies directed against pSTAT3 (yellow) (F). Arrows in C indicate CD31-positive microvessels. Cell nuclei were counterstained with DRAQ5 (blue); Scale bars: 25 μm. (A–E and G) Original magnification, ×200. (H) TNF-α secretion by ex vivo–cultured punch biopsies harvested from the colon of untreated and DSS-treated WT (black bars) and Hpa Tg (gray bars) mice on day 80 of the experiment. ND, non-detectable. (I) Increased levels of Tnfa mRNA determined by qRT-PCR in Hpa Tg (gray bars) versus WT (black bars) colon on day 80 of the experiment. Tnfa mRNA levels were normalized to actin mRNA (n = 5). (J) Microvessel counts in colons of Hpa Tg (gray bars) and WT (black bars) mice (n = 6). (K) Number of cyclin D1–positive cells in colons of Hpa Tg (gray bars) and WT (black bars) mice (n = 6). *P < 0.05, ***P < 0.001.
In addition to histopathological evaluation, we examined differences in chronic inflammatory phenotype between Hpa Tg and WT mice at the molecular level. Since TNF-α is an important player in the inflammatory cascade in both human UC and DSS experimental colitis, we first analyzed TNF-α production, applying ex vivo–cultured punch biopsies harvested from the colon of DSS-treated mice on day 80 of the experiment. At this stage, the amounts of TNF-α secreted by WT colonic explants were below the level detectable by ELISA (similar to explants harvested from healthy untreated colon), while 30.0 ± 6.1 pg/ml TNF-α were measured in medium conditioned ex vivo by colonic explants harvested from Hpa Tg mice (Figure 3H). In agreement, increased levels of mRNA encoding for TNF-α were determined by real-time PCR in Hpa Tg versus WT colons (Figure 3I).
TNF-α is a major inducer of NF-κB activation, and enhanced NF-κB signaling is the hallmark of chronic intestinal inflammation (18, 20). We therefore compared activation of the NF-κB pathway between WT and Hpa Tg mice. NF-κB is normally sequestered in the cytoplasm by means of association with an inhibitory protein, IκBα. Activation of NF-κB involves stimulation of the IKK complex which phosphorylates IκBα, triggering its degradation and nuclear translocation of active NF-κB (18). To examine NF-κB signaling, we analyzed phosphorylation of IκBα in colonic tissue derived from WT and Hpa Tg mice on day 80 of the experiment, immunostaining with an anti–phospho-IκBα (pIκBα) antibody. As shown in Figure 3D, a high number of pIκBα-positive cells was observed in Hpa Tg compared with WT colon. Cells positive for pIκBα were found in the stroma, and to a lesser extent in the epithelial compartment of Hpa Tg colon. Activation of NF-κB signaling was further confirmed by increased number of cells positive for nuclear phospho-p65 NF-κB, detected by immunofluorescent analysis of Hpa Tg versus WT colon (Supplemental Figure 3). Consistent with these findings, markedly increased levels of several NF-κB–inducible cytokines (36) (i.e., IL-1, IL-6, macrophage inflammatory protein-1 [MIP-1], and MIP-2) tightly associated with chronic colitis and/or the associated tumorigenesis (22, 37–41), were detected in Hpa Tg versus WT colon, applying mouse cytokine antibody array (data not shown) and confirmed by quantitative real-time PCR (qRT-PCR) (Supplemental Figure 4B and data not shown). In addition, increased expression of the NF-κB–regulated genes Bcl2l1 (Supplemental Figure 4B) and Cox2 (Figure 3E) was detected in Hpa Tg colonic tissue.
Notably, some NF-κB–controlled cytokines that were upregulated in Hpa Tg colon, including IL-1, IL-6, and TNF-α, play a pivotal role in activation of STAT3, a critical modulator of tumor-promoting chronic inflammation (42–45). We therefore assessed the activation status of STAT3 in our system, by immunostaining with anti–phospho-STAT3 (pSTAT3) antibody. As shown in Figure 3F, a high number of cells positive for nuclear localized pSTAT3 was observed in Hpa Tg as compared with WT colon (primarily in the epithelial compartment). In agreement with the recently established role of STAT3 in enhanced intestinal epithelial cell proliferation during colitis-associated tumorigenesis (42, 43), immunostaining with anti-PCNA antibody revealed an increased rate of proliferation of epithelial cells in Hpa Tg colon (Supplemental Figure 4A). Moreover, applying double-immunofluorescent staining with antibodies directed against PCNA and pSTAT3, we demonstrated that STAT activation colocalizes with highly proliferating epithelial cells in Hpa Tg colon (Supplemental Figure 4C). A significant increase in the rate of colonic epithelial cell proliferation in Hpa Tg versus WT mice was further confirmed by immunostaining with anti–cyclin D1 (Figure 3, G and K), providing a mechanistic explanation for the enhanced tumorigenesis observed in Hpa Tg mice (Figure 2, G and H). Of note, analysis of the apoptotic rate by immunostaining with antibody directed against cleaved caspase-3 revealed no statistically significant difference between Hpa Tg and WT mice in the percentage of cleaved caspase-3–positive cells (data not shown).
Taken together, these results suggest that the constantly elevated levels of heparanase in Hpa Tg mice (which resemble the observations in UC patients illustrated in Figure 1A) markedly affect the chronic phase of DSS-induced colitis and create a tumor-promoting inflammatory microenvironment via increased vascularization as well as enhanced NF-κB signaling, augmented levels of STAT3-inducing cytokines (i.e., IL-6, IL-1, TNF-α), and STAT 3 activation.
Macrophages represent both cellular targets for heparanase action and regulators of heparanase expression and activity in chronic colitis
Involvement of heparanase in macrophage activation. The chronic inflammatory microenvironment is characterized by infiltration with mononuclear cells, and macrophages are the dominant cellular players in chronic inflammation (32, 33). Lamina propria macrophages represent a major cellular source of TNF-α in IBD and play a key role in the pathogenesis of both human UC (46) and murine chronic colitis (47, 48). Additionally, macrophages are important contributors to tumor initiation and progression (33, 49). This effect is mediated, among other mechanisms, by the production of angiogenic and growth-promoting factors (i.e., VEGF, bFGF, HGF, EGF), cytokines (i.e., TNF-α) and enzymes (i.e., COX-2) (33, 49). This led us to examine the involvement of macrophages in our system. Through immunostaining with antibody directed against F4/80 (a mouse macrophage-specific marker; ref. 50), we detected increased macrophage infiltration in the colon of Hpa Tg versus WT mice on day 80 of the chronic DSS colitis model (Figure 4A). Of note, increased macrophage infiltration was also found in colonic tumors observed on day 110 of the AOM/DSS model in Hpa Tg versus WT mice (Supplemental Figure 5A). Macrophages infiltrating the Hpa Tg colon were not only more abundant than in WT colon, but also exhibited an exacerbated pro-inflammatory phenotype, as evidenced by a 3-fold increase (Figure 4C) in percentage of TNF-α–positive macrophages visualized by double immunofluorescent staining with anti–TNF-α and anti-F4/80 antibodies (Figure 4B). In support of this notion, double immunofluorescent staining with anti-pIκBα and anti-F4/80 antibodies showed increased NF-κB signaling in macrophages infiltrating the Hpa Tg colon (Supplemental Figure 5B). These findings led us to assume that heparanase overexpression directly affects macrophage recruitment and activation. LPS derived from Gram-negative bacteria is a specific stimulator of TLR4 signaling (51) and is widely used in macrophage activation studies. In fact, TLR-4–mediated activation of macrophages is recognized as a key event in chronic colitis and the associated tumorigenesis (52, 53). To validate the role of heparanase in macrophage activation, we isolated mouse peritoneal macrophages and stimulated them with LPS in the absence or presence of recombinant active heparanase to recapitulate conditions that occur in UC (i.e., heparanase-rich environment and abundant microbial flora). Pretreatment with heparanase strongly sensitized macrophages to activation by LPS, as indicated by a 7-fold increase in TNF-α secretion (Figure 4D) and by a significant increase in mRNA of TNF-α as well as IL-6 and IL-12p35 (Figure 4E), two additional macrophage-derived cytokines known to be induced by TLR4 signaling and tightly involved in the pathogenesis of UC (54). Notably, sensitization of macrophages to LPS by heparanase was dependent on its enzymatic activity, since pretreatment with heat-inactivated heparanase (iHpa) did not affect macrophage activation by LPS (Figure 4, D and E). These findings are in agreement with previous reports showing that extracellular HS in its intact form inhibits LPS-mediated TLR4 signaling and macrophage activation and that its removal relieves this inhibition (55). To confirm that heparanase activity is capable of decreasing cell surface HS in this system, we compared the bFGF binding capacity of mouse macrophage-like RAW264.7 cells untreated or treated with heparanase, as previously described (26), taking into account that HS functions as a low-affinity/high-capacity receptor for bFGF (56). Heparanase-treated and -untreated RAW264.7 cells were incubated with increasing concentrations of 125I-bFGF, and their ability to bind bFGF was evaluated by measuring the radioactivity associated with the cells. Under the applied experimental conditions, bFGF binds primarily to HS (26). As demonstrated in Figure 4F, binding of 125I-bFGF to heparanase-treated cells was markedly decreased, indicating that heparanase reduces the amount of HS on the macrophage cell surface. Taken together, the above data suggest that upregulated heparanase enables enhanced activation of macrophages, reprogramming their response from resolution of inflammation to unresolved chronic colitis.
Effect of heparanase on macrophages. (A) Increased macrophage accumulation during chronic DSS colitis in Hpa Tg colon. Colonic tissue was obtained from Hpa Tg and WT mice on day 80 and stained with anti-F4/80 antibody. F4/80-positive macrophages were quantified per 100-μm2 microscopic field, based on 6 sections from 3 independent mice of each group. Inset: F4/80-positive macrophages (yellow) infiltrating WT (top) and Hpa Tg (bottom) colon. Scale bars: 50 μm. (B) Increased number of TNF-α–expressing macrophages in Hpa Tg colon. Colonic tissue was processed for double immunofluorescent analysis with anti-F4/80 (yellow) and anti–TNF-α (red) antibodies. Cell nuclei were counterstained with DRAQ5 (blue). Scale bars: 50 μm. Insets: Enlarged images of the area delineated by dashed lines. Orange arrows indicate TNF-α–expressing macrophages; white arrowhead indicates TNF-α–negative macrophage. Scale bars: 5 μm. (C) F4/80 and TNF-α double-positive cells (TNF-α–expressing macrophages) were quantified per 100-μm2 microscopic field (6 sections from 3 independent mice), and the percentage of TNF-α–positive macrophages was calculated. (D and E) Heparanase sensitizes macrophages to LPS activation in vitro. Mouse peritoneal macrophages were untreated (Cont) or treated (2 hours, 37°C) with active recombinant heparanase (0.8 mg/ml), LPS (100 ng/ml), heparanase followed by LPS (Hpa+LPS), heat-inactivated heparanase (iHpa) alone, or iHpa followed by LPS (iHpa+LPS). (D) TNF-α secretion was evaluated by ELISA. (E) TNF-α, IL-6, and IL-12p35 expression was assessed by qRT-PCR. (F) Heparanase decreased HS content on macrophage cell surface. RAW264.7 macrophages were either untreated (Cont) or treated (2 hours, 37°C) with active heparanase, and HS content was assessed as described in Methods. ***P < 0.001.
Although heparanase enzymatic activity appears to play a central role in this phenomenon, it should be noted that under certain experimental conditions heparanase may also exert enzymatic-independent effects, the best documented of which is induction of Akt/PKB phosphorylation (3). In light of the importance of Akt/PKB signaling in macrophage function (57–59), we tested whether the effect of heparanase on Akt phosphorylation may be responsible (at least in part) for the observed sensitization of macrophages in our system. Applying immunoblotting with anti–phospho-Akt (pAkt) antibody, we compared activation levels of Akt in macrophages untreated or treated with heparanase in the absence or presence of LPS. Lack of increase in pAkt following treatment with heparanase alone, as opposed to 3.5 fold increase in pAkt levels detected in macrophages exposed to LPS in either the absence (in agreement with ref. 58) or presence of heparanase (data not shown) suggests that non-enzymatic activity (i.e., induction of Akt/PKB signaling) is less relevant in heparanase-mediated sensitization of macrophages. However, the contribution of additional, currently unknown non-enzymatic effects of heparanase could not be excluded.
Induction of heparanase expression by activated macrophages. In light of the ability of inflammatory cytokines to induce heparanase expression (60), it is plausible that activated macrophages represent an important source of cytokines capable of stimulating overexpression of epithelial heparanase in colitis. In support of this assumption, incubation of Colo205 colon carcinoma cells with medium conditioned by LPS-activated macrophages resulted in a more than 3-fold increase in heparanase levels compared with Colo205 cells that remained untreated or incubated with either LPS alone, or medium conditioned by resting macrophages (data not shown). Based on reports implicating TNF-α in upregulation of heparanase in endothelial cells (60), we hypothesized that macrophage-derived TNF-α is a likely mediator of heparanase induction in this system. In subsequent experiments, Colo205 cells were maintained untreated or treated with TNF-α for 24 hours and heparanase expression was assessed by real-time PCR, immunofluorescent staining, and activity assay. As shown in Figure 5, A–C, TNF-α treatment caused a marked increase in heparanase mRNA levels, protein content, and enzymatic activity. Similar results were obtained utilizing the Caco-2 colon carcinoma cell line (data not shown). In addition, the ability of TNF-α to upregulate heparanase in normal colonic epithelium was demonstrated by increased heparanase expression in colonic explants (derived from healthy mouse colon) cultured ex vivo in the presence of TNF-α (15 ng/ml; Figure 5, D and E), and by induction of heparanase in colonic epithelium of healthy mice following i.p. injection of TNF-α (Figure 5F). Next we tested the effect of the TNF-α antagonist etanercept, previously shown to inhibit bioactivity of murine TNF-α (61), on heparanase production in mouse colon during DSS colitis. For this purpose, mice receiving 5% DSS in the drinking water for 7 days were injected i.p. with either vehicle (saline) alone or etanercept (3 mg/kg body weight, twice a day, starting on experimental day 1). On experimental day 8, mice were weighed and sacrificed, and heparanase expression was assessed by immunostaining and enzymatic activity determined in medium conditioned ex vivo by colonic explants harvested from etanercept-treated and -untreated mice. As shown in Supplemental Figure 6, A and B, etanercept treatment markedly reduced heparanase induction upon DSS challenge, further confirming the role of TNF-α in heparanase upregulation in vivo.
TNF-α induces heparanase expression in Colo205 colonic carcinoma cells and in normal mouse colonic epithelium ex vivo and in vivo. (A–C) Colo205 cells were incubated (16 hours) in triplicate in the absence or presence of 15 ng/ml TNF-α. Heparanase expression was assessed 16 hours later by (A) immunofluorescent staining (scale bars: 20 μm), (B) qRT-PCR determination of mRNA levels, and (C) enzymatic activity assay. (D and E) Heparanase induction in colonic explants treated with TNF-α ex vivo. Explants harvested from healthy mouse colon as described in Methods were cultured ex vivo for 16 hours in the absence and presence of TNF-α (15 ng/ml). Heparanase expression was assessed 16 hours later by (D) qRT-PCR determination of mRNA levels and (E) immunofluorescent (yellow) staining (scale bars: 50 μm). (F) Induction of heparanase expression in mouse colonic epithelium following TNF-α administration in vivo. Colonic tissue specimens derived from healthy BALB/c mice injected i.p. with PBS or TNF-α (1 μg/mouse, 24 hours) were stained with anti-heparanase antibody (brown staining). Note heparanase staining in colon epithelium derived from TNF-α–treated but not PBS-treated mice (original magnification, ×200).
Role of macrophages in proteolytic activation of heparanase. During the course of DSS-induced colitis in WT mice, we observed not only an increase in total heparanase expression, but also augmented levels of proteolytically processed active heparanase (Figure 1, D–F). In accordance, increased levels of CatL mRNA and protein (Figure 1, C and D) were observed in extracts of mouse colonic tissues at corresponding time points. However, no notable CatL expression was detected by immunostaining in heparanase-overexpressing inflamed colonic epithelium during any stage of DSS colitis (Figure 1G). Instead, CatL-positive cells (primarily immunocytes) were accumulated in the stromal compartment of the colon during all stages of chronic colitis (Figure 1G). It has been previously shown that CatL plays an important pathophysiological role in colonic inflammation and that macrophages are the primary cellular source of inducible CatL expression in inflamed colon of UC patients and in acute DSS-induced mouse colitis (62). A different cellular source of heparanase (epithelial) and CatL (stroma/immunocytes) implies that proteolytic processing of heparanase by CatL takes place in the extracellular space. The secreted nature of heparanase in our system was demonstrated by the presence of heparanase enzymatic activity in medium conditioned by ex vivo–cultured punch biopsies harvested from the colon of DSS-treated versus untreated mice on day 59 of the experiment (Figure 1F). While several cell types may secrete inactive proforms of CatL, activated macrophages are unique in their ability to secrete mature CatL through a specific mechanism that stabilizes CatL and allows extracellular accumulation of the active enzyme (63). Altogether, these data led us to hypothesize that in chronic colitis, activated macrophages are decisively involved in proteolytic processing and activation of the latent proheparanase, providing a pool of extracellular active CatL. To test this hypothesis, we first applied double-immunofluorescent staining with antibodies directed against the macrophage marker F4/80 and CatL in mouse DSS colitis samples. As shown in Figure 6A, the majority of F4/80-positive cells in WT colon specimens harvested on day 59 of DSS colitis were also positive for CatL, resembling the findings reported in inflamed mucosal tissue of UC patients (62). To test whether activated macrophages are capable of proteolytic processing and activation of pro-heparanase, we incubated purified 65-kDa heparanase precursor with the conditioned medium of resting or LPS-stimulated mouse peritoneal macrophages, in the absence or presence of specific and irreversible CatL inhibitor and examined the resulting heparanase enzymatic activity. As shown in Figure 6B, incubation of the 65-kDa heparanase precursor with medium conditioned by resting macrophages resulted in little or no heparanase activity. By contrast, heparanase processing and activation became evident upon incubation of the 65-kDa precursor with medium conditioned by LPS-stimulated macrophages (Figure 6B). Moreover, the increase in heparanase activity was abolished in a dose-dependent manner in the presence of the specific irreversible CatL inhibitor (Figure 6B). Similar results were obtained when macrophages were activated with IFN-γ instead of LPS (data not shown). Collectively, these findings suggest the occurrence of a vicious cycle through which heparanase stimulates macrophage activation, while the activated macrophages stimulate further production and activation of heparanase.
Role of macrophage-derived CatL in proteolytic activation of heparanase. (A) Colonic tissue was harvested on day 59 and processed for double immunofluorescent analysis applying anti-F4/80 and anti-CatL antibodies. Note that the vast majority of CatL-producing cells are F4/80-positive macrophages. (B) Proteolytic processing and activation of latent heparanase by macrophages. Purified 65-kDa heparanase precursor (proHpa) was incubated with medium conditioned by resting (rMϕ) or LPS-stimulated (sMϕ) mouse peritoneal macrophages. Heparanase enzymatic activity was examined using sulfate-labeled ECM as substrate. Note lack of activity following incubation of the 65-kDa precursor with medium conditioned by rMϕ, as compared with readily detectable activity upon incubation with medium conditioned by sMf. Inset: Proheparanase was incubated with medium conditioned by sMϕ in the absence or presence of increasing concentrations of specific CatL inhibitor I (compound Z-Phe-Phe-CH2F; Calbiochem catalog no. 421419). Heparanase enzymatic activity was examined using sulfate-labeled ECM as substrate.