Specific inhibition of plasma kallikrein modulates chronic granulomatous intestinal and systemic inflammation in genetically susceptible rats

^a Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA
^b Center for Gastrointestinal Biology and Diseases; University of North Carolina, Chapel Hill, North Carolina 27599, USA
^c DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880–0500, USA
^d Department of Pharmacy, University of Montreal, Montreal, Quebec H3C 3J7, Canada

	ABSTRACT

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The kallikrein-kinin (K-K) (contact) system is activated during acute and chronic relapsing phases of enterocolitis induced in genetically susceptible Lewis rats by intramural injection of peptidoglycan-polysaccharide (PG-APS). Using the selective plasma kallikrein inhibitor P8720, we investigate whether activation of the K-K system plays a primary role in chronic granulomatous intestinal and systemic inflammation in this model. Group I (negative control) received human serum albumin intramurally. Group II (treatment) received PG-APS intramurally and P8720 orally. Group III (positive control) received PG-APS intramurally and albumin orally. P8720 attenuated the consumption of the contact proteins, high molecular weight kininogen (P<0.03), and factor XI (P<0.04) in group II vs. group III. P8720 decreased chronic intestinal inflammation measured by blinded gross (P<0.01) and histologic (P<0.0005) scores as well as systemic complications (arthritis, splenomegaly, hepatomegaly, leukocytosis, and acute-phase reaction) (P<0.01) in group II as compared with group III. We conclude that relapsing chronic enterocolitis and systemic complications are in part due to plasma K-K system activation, and that inhibition of this pathway is a potential therapeutic approach to human inflammatory bowel disease and associated extraintestinal manifestations.—Stadnicki, A., Sartor, R. B., Janardham, R., Majluf-Cruz, A., Kettner, C. A., Adam, A. A., Colman, R. W. Specific inhibition of plasma kallikrein modulates chronic granulomatous intestinal and systemic inflammation in genetically susceptible rats. FASEB J. 12, 325–333 (1998)

Key Words: kininogen • chronic enterocolitis • arthritis • protease inhibitor

	INTRODUCTION

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THE COURSE OF the inflammatory bowel diseases (IBD),² such as ulcerative colitis and Crohn's disease, is characterized by local and systemic inflammation as well as unpredictable relapses and remissions (1). Although these disorders are idiopathic, they appear to be immunologically mediated and have a genetic component (1, 2). In animal models, the aggressiveness and chronicity of the inflammatory process is dependent on the genetic background of the host (2, 3). We have developed an experimental model of chronic granulomatous enterocolitis that permits detailed examination of the mechanisms of genetically determined intestinal and systemic inflammation (4). We use a bacterial cell wall polymer, peptidoglycan-polysaccharide, from group A streptococci (PG-APS) (5), injected intramurally to induce intestinal inflammation. Acute inflammation develops at the injected area of the intestine in all rat strains investigated, but the spontaneous reactivation of chronic granulomatous enterocolitis is restricted to genetically susceptible Lewis and Sprague-Dawley rats and does not appear in Buffalo and Fisher rats (4, 6, 7). Female Lewis rats, the highest responders, develop acute intestinal inflammation that peaks 1–2 days after PG-APS injection, gradually decreases over the next 10 days, and spontaneously reactivates beginning on day 14, accompanied by peripheral erosive arthritis, granulomatous hepatitis, normochromic anemia, and leukocytosis (4), with histological findings of fibrosis and granulomas resembling Crohn's disease.

Inflammation induced by PG-APS, similar to human IBD, is mediated by a panoply of inflammatory cascades. This bacterial product has been shown to stimulate synthesis of cytokines, eicosanoids, nitric oxide, oxygen radicals, and proteases by inflammatory effector cells as well as to activate the coagulation, complement, and fibrinolytic cascades (5, 8, 9). We have recently demonstrated activation of the kallikrein-kinin (K-K) (contact) system during acute and chronic phases of intestinal inflammation in Lewis rats, but not in Buffalo rats, although both rat strains display acute inflammation (4). Moreover, our most recent data showing contact pathway activation in indomethacin-induced enterocolitis in Lewis rats (10) indicate that activation of this pathway is not restricted to inflammation induced by exogenous bacterial products. Negatively charged surfaces such as endotoxin (11), activated blood, or endothelial cells are able to autoactivate zymogen factor XII (FXII), the initial component of the contact system, to its active form, FXIIa. This enzyme converts prekallikrein (PK) to kallikrein and coagulation factor XI (FXI) to FXIa, the initiating component of intrinsic coagulation. Kallikrein in the presence of high molecular weight kininogen stimulates neutrophil chemotaxis (12), aggregation (13), and oxygen consumption (14), and induces those cells to release elastase (15). FXIIa is also an agonist for neutrophils (16) and monocytes (17), and initiates the classical complement cascade (18). Kallikrein stimulates the cell-bound fibrinolytic system by converting prourokinase to urokinase (19) and activates the alternative complement pathway (20). Moreover, kallikrein amplifies contact activation by catalyzing the conversion of FXII to FXIIa, which hydrolyzes PK to kallikrein and the conversion of FXI to FXIa (21). Kallikrein also cleaves high molecular weight kininogen (HK) to produce bradykinin, which stimulates constitutive B2 and inducible B1 receptors on endothelial cells, smooth muscle cells, epithelial cells, and fibroblasts (22). This potent inflammatory peptide enhances vasodilation, increases vascular permeability, and influences intestinal motility and electrolyte secretion (22, 23).

We have recently developed a peptide derivative of arginine boronic acid (P8720), which is a specific tight-binding reversible inhibitor of plasma kallikrein (24). Based on its affinity and selectivity in the inhibition of plasma kallikrein, P8720 was tested for efficacy in vivo. We successfully used this specific inhibitor to modulate acute arthritis (24) as well as acute enterocolitis (8) induced by PG-APS in Lewis rats, but P8720 has not yet been tested in the chronic T lymphocyte-dependent phase of this model. Chronic granulomatous entercolitis and associated systemic inflammation display more unique and characteristic morphological, clinical, and genetic features that reinforce its relevance to Crohn's disease.

In the present study, chronic inflammation was treated orally with P8720. We measured the pathophysiological changes of chronic intestinal and systemic inflammation by blinded gross and histologic scoring. The systemic acute-phase response was assayed by measurement of plasma T-kininogen concentration, a positive type 2 acute-phase protein in rats. To ascertain the kallikrein-kinin and coagulation system responses, we performed quantitative functional assays of PK, FXI, HK, and antithrombin III (ATIII). We demonstrate that this selective plasma kallikrein inhibitor modulates reactivation of intestinal and systemic inflammation by preventing activation of the contact system.

	MATERIALS AND METHODS

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Inhibitor of plasma kallikrein
The peptide, a boroarginine derivative of boronic acid Bz-Pro-Phe-boroArg-OH (P8720, DuPont Merck), was synthesized as described in U.S. Patent 5,187,157. The properties of this inhibitor were described in our previous study (24). The K_i for plasma kallikrein was 0.15 nM, an affinity 100-fold greater than that of plasmin (K_i=16 nM) and 300-fold higher than those of thrombin or tissue (pancreatic) kallikrein (K_i=42 nM). All other plasma serine proteases and complement active enzymes displayed a K_i at least 500-fold less than that of kallikrein.

PG-APS polymers
Purified, sterile PG-PS fragments from the cell walls of group A, type 3, strain D58 streptococci (Streptococcus pyogenes) were prepared as described previously (4).

Experimental protocol and treatment
A total of 21 female inbred specific pathogen-free Lewis rats (Charles River Laboratories, Raleigh, N.C.), mean weight 158 g, were used. Rats were divided into three different groups. Group I (negative control, n=5) was injected subserosally with human serum albumin (HSA), 37.5 mg/kg body weight, intramurally in the intestine as described (4), and treated orally with 0.1% HSA every 8 h beginning 2 h before HSA injection. Group II (treatment, n=8) was injected intramurally in the intestine with PG-APS, 12.5 mg rhamnose/kg body weight, and treated orally with P8720 (21 mg/kg, every 8 h for 18 days delivered in 0.3 ml of 0.1% HSA by gavage with a feeding needle). Group III (positive control, n=8) received 12.5 mg rhamnose/kg body weight of PG-APS and was treated orally every 8 h with 0.1% HSA.

Intestines were exposed by laparotomy using intramuscular anesthesia with 80 mg/kg ketamine, 1.5 mg/kg acepromazine, and an aseptic technique, and injected subserosally with either PG-APS or HSA into seven locations of the distal ileum and cecum, as previously described (4). Animals were killed with carbon dioxide narcosis at 18 days after surgery.

Quantification of intestinal inflammation
Intestinal inflammation was quantified by gross and histologic methods. At necropsy, performed by a blinded observer, a gross gut score was calculated using the sum of 0–4 scores of four independent parameters, including: intestinal wall thickening, adhesions, mesenteric contraction, and serosal nodules (granulomas) as previously described and validated (6). Thus, the maximum score was 16. Samples from the Peyer's patches, distal ileum, and cecum were fixed in 10% buffered formalin for histological processing. Blinded histological assessment of acute and chronic inflammation was performed as previously described and validated (4, 7). Briefly, components of the acute inflammatory score consisted of polymorphonuclear leukocytic infiltration, edema, and necrosis, whereas a chronic inflammatory score was based on the number of mononuclear cells (macrophages and lymphocytes). Values 0–4 were noted for acute and chronic inflammatory cell infiltrations at each site. Acute and chronic scores were assigned for each of the four sites (Peyer's patches, distal ileum, cecal tip, and midcecum). The presence of crypt abscesses and granulomas was noted. The acute and chronic scores for each section were totaled; thus, the maximum possible total histological score was 32.

Systemic inflammation
Rats were weighed prior to surgery and every 2 days for the duration of the experiment. Arthritis was serially quantitated using rear ankle joint diameters measured with digital calipers (Fowler UltraCal II, Sylvac). A mean of duplicate measurements of each rear ankle was recorded from 10 to 18 days after PG-APS injection. At necropsy, liver and spleen weights were recorded. Hepatic granulomas were blindly quantified on a 0–4 scale. Cardiac blood was collected for hematologic measurements (white blood cell count and hematocrit) and plasma coagulation protein analysis as described previously (4).

Assays of components of kallikrein-kinin system and anti-thrombin
Plasma prekallikrein functional levels were performed by a microtiter, amidolytic assay by using the chromogenic substrate, S-2302 (Pharmacia, Franklin, Ohio) (25). Using a mixture of activated factor XII, HK, and ellagic acid in inhibitor-depleted plasma, the assay activates more than 90% of prekallikrein to kallikrein. Factor FXI functional levels were determined by measuring the hydrolysis of the chromogenic substrate S-2366 (Pharmacia) by activated FXI generated in the assay (26). We used kaolin as an activator of factor XIIa in inhibitor-depleted plasma in the presence of soybean trypsin inhibitor (to prevent plasma kallikrein activity) and corn trypsin inhibitor (to prevent hydrolysis of substrate by factor XIIa). More than 95% of factor XI is converted to activated factor XI. High molecular weight kininogen activity was evaluated according to a modification of the partial thromboplastin time assay described by Proctor and Rapaport (27), using total kininogen-deficient plasma (28). Antithrombin was measured by a functional amidolytic assay (Coatest Antithrombin Kit, Helena Laboratories, Beaumont, Tex.) (29). We previously studied the influence of P8720 on each of the contact protein assays over a range of concentration of 48–392 nM. No effect was found on the FXI, HK, or antithrombin. However, the functional levels of PK were altered by P8720 (8).

T-kininogen determination
Plasma T-kininogen was measured by sandwich enzyme-linked immunosorbent assay, as described previously (3, 30).

Measurement of glutamic pyruvic transaminase in rat plasma
The activity of glutamic pyruvic transaminase (GPT) in rat plasma was measured with a dimensional clinical analyzer (DuPont, Wilmington, Del.) according to the method of Bergmeyer et al. (31).

Measurement of P8720 inhibitor concentration
The functional concentration of P8720 in rat plasma was determined, by a method developed in this laboratory (8, 24), from its ability to inhibit purified plasma kallikrein (36 nM).

Statistical analysis
Plasma from 10 female Lewis rats that received neither saline nor PG-APS was used to derive normal mean values of the assays with which to normalize the data from each group. Differences among groups were analyzed by the two-way analysis of variance method of Newman-Keuls to validate the data (32). The unpaired Student's t test was then used to evaluate the difference among groups. P values <0.05 were considered significant.

	RESULTS

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Concentration of P8720 in rat plasma
The mean plasma level of P8720 in the treated rats, group II (n=8), was 58 ± 2.9 nM 2–4 h after oral administration of 21 mg/kg, which yielded a 66 ± 3.0% inhibition of purified kallikrein (36 nM) in the in vitro assay.

Chronic intestinal inflammation
Eighteen days after PG-APS injection, PG-APS-injected Lewis rats treated with HSA (positive controls) displayed chronic intestinal inflammation with thickening of the intestinal wall, dense adhesions, and serosal nodules. Inflammation occurred in the area of injection and was especially severe in the cecum and distal ileal mesentery. The PG-APS-injected, P8720-treated rats showed a decrease in all components of the gross inflammatory score, cecal wall thickening, intestinal contraction, adhesions, and (of particular interest) serosal nodules (0.9±0.2, P8720-treated vs. 2.1±0.3, positive control, P<0.001). The control rats injected with HSA had no gross inflammatory changes. The mean gross gut score was significantly higher (P<0.001) in group III, positive control (9.8±1.2), and group II, treatment (4.6±0.9), when compared with group I, negative control (0±0). However, the gross gut score was significantly lower after P8720 treatment (P<0.01) in group II was compared with group III ( Fig. 1A). Histological assessment of acute and chronic components of inflammation clearly confirmed the gross observations. Lewis rats injected with PG-APS showed transmural granulomatous inflammation with extensive fibrosis. Predominant infiltrating cells in the mucosa and submucosa were mononuclear cells and neutrophils. Crypt abscesses were present in the mucosa; granulomas in the lamina propria were found in typical rats from group III ( Fig. 2A). In contrast, mild inflammation was seen in rats in group II treated with P8720, with less infiltration by neutrophils and only minimal fibrosis ( Fig. 2B). No changes were observed in negative control rats from group I. The rats treated with P8720 experienced a significant decrease (P<0.005) in the acute histological score, a marker of granulocyte infiltration, when compared with group III (5.1±1.3 vs. 10.1 ± 0.5) ( Fig. 1B). Negative controls (group 1) had no detectable acute inflammation (0±0). Similarly, the intestinal total histological score was significantly higher in group III (20.9±0.8) than group II (12.6±2.2, P<0.005) or group I (0.2±0.1, P<0.0001) ( Fig. 1C). Thus, treatment with the kallikrein inhibitor P8720 markedly attenuated the spontaneously reactivating granulomatous phase of intestinal inflammation.

View larger version (18K): [in this window] [in a new window]   Figure 1. Gross and histological changes in intestinal inflammation. A) Gross inflammation. B) Acute histological inflammation. C) Total histological inflammation. Blinded scores were calculated as described in Materials and Methods. Solid bars indicate group I (negative control), open bars group II (treatment), and hatched bars group III (positive control) *P < 0.01, **P < 0.005, group II vs. group III. Values are expressed as mean ±SEM.

View larger version (286K): [in this window] [in a new window]   Figure 2. Histological alterations in rat intestine at 18 days. A) Granulamatous inflammation in a representative rat cecum from group III (positive control) with transmural colitis, granulomas (arrow) and fibrosis. B) Histological appearance of rat cecum from group II (treated group) showing minimal inflammation with no architectural changes and no acute neutrophilic infiltration. H & E, original magnification: x25.

Extraintestinal complications
Development of chronic arthritis in group III, positive control, was indicated by a progressive increase in ankle joint diameter from day 10 to day 18 ( Fig. 3). In contrast, the rats in group II, treated with kallikrein inhibitor P8720, experienced a pronounced attenuation of joint enlargement as compared with group III at all measured time points (83% at days 17 and 18). As shown in Table 1, liver weight was highest in group III (P<0.001 vs. group I), but was significantly decreased by treatment with P8720 (P<0.01 vs. group III). Similarly, spleen weight was found to be significantly lower (P<0.05) after kallikrein blockade. The rats in group III displayed a significant increase in peripheral blood white cell count (P<0.0001 vs. group I); however, P8720 significantly inhibited this increase in group II (P<0.005 vs. group III). The white cell count in group II was significantly higher (P<0.001) when compared with group I. The hematocrit value was lowest in group III (35.5±1.8%); P8720 in part attenuated this decrease in group II (39.8±1.1), but the difference between groups did not reach statistical significance. Similarly, the decreased grossly evident liver granulomas after kallikrein inhibition was not statistically significant. Thus, therapy with P8720 almost completely prevented the development of arthritis and markedly attenuated liver/spleen enlargement and leukocytosis associated with chronic intestinal inflammation.

View larger version (17K): [in this window] [in a new window]   Figure 3. Effect of P8720 therapy on progressive arthritis accompanying intestinal inflammation in Lewis rats. Changes in joint diameter. Shown are serial determinations at 10, 14, 15, 16, 17, and 18 days in group III, positive control (closed circles), group II, treatment (open circles), and group I, negative control (closed triangles). See Fig. 3. *P < 0.05; **P < 0.005; < 0.0001 group III vs. group II.

Toxic effects of P8720
In seven of eight rats in group II treated with P8720, the plasma levels of the GPT were within normal ranges (normal upper limit = 80 units/l), indicating no evidence of hepatotoxicity. However, in one of those rats, the plasma GPT levels were found to be markedly increased (674 units/l). Nevertheless, physiological and biochemical parameters, including ATIII concentration (a sensitive marker of liver protein synthesis), for this rat were not much different from those of the remaining animals. Transaminase values of group III (PG-APS, positive control) were normal despite the presence of hepatomegaly and granulomas. We also compared weight changes in the three groups of rats. The mean weight of rats before the experiment (day 0) in group I (159±0.7 g), group II (158±0.7 g), and group III (159±2 g) was nearly equal. Rats in group I displayed progressive weight gain. At the beginning of the experiment, rats in group II and group III displayed weight loss, which was more pronounced in group II (treated with P8720) than in group III (136±2.0 g vs. 150±1.5, P<0.01, at day 2). At day 17, the mean weights of rats in groups I (193±2.7) and III (188±2.2) were not significantly different. However, rats treated with P8720 weighed less (170±4.2, <0.01) compared with groups I and II. The lower weight gain in rats of group II during the experiment indicates drug toxicity.

Biochemical assays of contact and coagulation proteins
As demonstrated in Fig. 4, the rats in group III, positive control, showed a significant fall (P<0.01) in plasma PK ( Fig. 4A), FXI ( Fig. 4B), and HK ( Fig. 4C) functional levels as compared with group I, negative control. The PK value in rats from group II, treated with P8720, does not reflect plasma functional PK level due to in vitro interference of P8720 with the PK assay. P8720 therapy significantly diminished the decrease of FXI (P<0.04) as well as HK (P<0.03) in group II as compared with group III. Moreover, there were no significant changes between FXI and HK levels in group II as compared the negative controls. Thus, treatment with the kallikrein inhibitor P8720 markedly prevented plasma K-K system activation in the treated group, as reflected in an attenuation of the decrease of HK and factor XI as compared with the positive control group. In contrast, the mean functional ATIII levels in the three groups examined did not differ significantly from each other (data not shown), indicating preservation of hepatic protein synthesis in all groups.

View larger version (18K): [in this window] [in a new window]   Figure 4. Biochemical assays of contact system proteins. This figure shows functional levels of rat plasma prekallikrein (A), factor XI (B), and high molecular weight kininogen (C). Solid bars represent group I, negative control; open bars group II, treatment; and hatched bars group III, positive control. Values are expressed as mean ±SEM.

Acute-phase protein
Plasma T-kininogen antigenic levels, an acute-phase protein in the rat, were significantly increased in group III (6.63±0.39 mg/ml) (P<0.001) and group II (4.06±0.6) (P<0.01) when compared with group I (0.63±0.09), as shown in Fig. 5. However, the T-kininogen level in group II was significantly lower (P<0.01) as compared with group III, which indicates that P8720 therapy decreased the systemic inflammatory response.

View larger version (40K): [in this window] [in a new window]   Figure 5. Biochemical assay of T-kininogen. This figure represents antigenic levels of plasma T-kininogen, a rat acute-phase reactant. Solid bar indicates group I, open bar group II, and hatched bar group III; mean ±SEM.

	DISCUSSION

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K-K system activation has been demonstrated in various experimental and human disease states caused by bacterial infection and/or trauma (21, 22). Thus, this system plays a role in inflammatory processes and in responses to tissue injury. However, the importance of the K-K system in regulating pathophysiological responses is most conclusively documented by specific inhibition. Implication of the K-K system in the pathogenesis of acute arthritis and experimental sepsis has been well documented by studies with specific inhibitors in our laboratory (24, 33). More recently, we have documented that a specific kallikrein inhibitor, P8720, attenuated the acute phase of PG-APS-induced intestinal inflammation in Lewis rats (8). However, acute entercolitis occurs not only in genetically susceptible inbred Lewis rats, but also in genetically resistant rat strains (Buffalo and Fisher); thus, the acute inflammatory response is relatively nonspecific in this model (4) and is mediated by different immunologic mechanisms than is the chronic granulomatous phase, which is T lymphocyte dependent (34).

In the present study, we assessed the role of the K-K system in the chronic, relapsing phase of intestinal and systemic inflammation by using the same highly effective, selective kallikrein inhibitor used in the acute studies (8, 24). Lewis rats (positive control group) analyzed 18 days after PG-APS injection displayed a pattern of K-K system activation similar to that of the acute phase (8), manifested by a decrease in the plasma concentration of contact proteins PK, FXI, and HK. Unchanged ATIII values indicate that low levels of the contact proteins are not related to its consumption during extrinsic coagulation or to decreased liver protein synthesis (8, 21). In the current study, oral administration of P8720 to treated rats resulted in a mean level of 58 nM, which resulted in a 66% inhibition of in vitro kallikrein activity. This percent of inhibition was lower than we observed in rats treated by P8720 in the acute phase in this model (87%), commensurate with the lower doses administered (21 mg/kg every 8 h vs. 18 mg/kg every 6 h in the acute study). However, this degree of inhibition is similar to that observed in our laboratory in FXII activity when using anti-FXII monoclonal antibody in baboon experimental sepsis (33). Despite incomplete inhibition of the contact system in both the sepsis model and the current experiment, we observed in both a remarkable attenuation of inflammatory responses as well as K-K system activation. Thus, 60–65% inhibition of this system seems to be sufficient in vivo to modulate inflammatory changes and their biochemical correlates. In the present study, plasma P8720 inhibitor partially interfered with plasma kallikrein activity measured by functional assay, as we also demonstrated previously, both in vitro and in vivo (8, 24). However, HK and FXI changes observed are consistent with the selective inhibition of kallikrein in vivo. The 30% decrease of plasma HK found in the positive control group was prevented considerably by treatment with P8720. Since kallikrein also amplifies contact activation by cleaving FXII to FXIIa and forms HKa (21), a cofactor necessary for activation of FXII, its specific inhibition attenuates the decrease of FXI.

Administration of this specific kallikrein inhibitor prevented in part the development of chronic intestinal and systemic inflammation in Lewis rats. The gross gut score was decreased far more (53%) than observed previously in the acute phase (27%), even though the present study used lower doses of the same P8720 inhibitor. We present here histological confirmation showing that P8720 suppressed the increased total intestinal histological score by 40% and the acute histological score by 50%. Since the acute histological score reflects mainly polymorphonuclear leukocytic infiltration, its impressive decrease corresponds well with the decrease in gross gut score and decrease of acute-phase reaction we demonstrated only in the acute arthritis model (24). Together, these results strongly indicate the importance of kallikrein as a neutrophil chemotactic enzyme and agonist in vivo. Based on in vitro studies, the specific kallikrein inhibitor should decrease intestinal neutrophil recruitment (12), release of lysosomal enzymes (16), and formation of toxic oxygen radicals (14). The effect of the specific kallikrein inhibitor to suppress enterocolitis may be due in part to prevention of bradykinin release.

The very early intestinal vascular events induced by PG-APS (35) and acute inflammation in other experimental colitis models are known to be related to eicosanoid production. Chronic granulomatous intestinal inflammation induced by PG-APS is far more complex and represents the true reactivation of enterocolitis rather than persistence of acute inflammation (3, 4). Reactivation is documented by higher gross and histological inflammatory scores during the chronic phase compared with the acute phase and abrupt onset of chronic enterocolitis and arthritis at 14 days after a quiescent phase (4, 6). Intramural and granulomatous intestinal inflammation with fibrosis and dense adhesions as a result of amplification of inflammatory cascades is mediated in part by cytokines such as interleukin-1 (IL-1) (6). The role of cytokines in PG-APS-induced enterocolitis has been investigated by Sartor and colleagues (6, 9). Inhibition studies have implicated IL-1 in the pathogenesis of acute and chronic enterocolitis in this model (6). Therapy directed toward inhibition of T lymphocyte function has demonstrated that chronic but not acute inflammation in this model is T lymphocyte dependent (34), presumably due to secretion of lymphokines such as interferon , which is up-regulated during chronic inflammation (9). Administration of neutralizing monoclonal antibodies against factor XII to baboons suffering from sepsis is accompanied by reduced IL-6 release (36). Tumor necrosis factor (TNF) and IL-1 may have indirect effects via other inflammatory cytokines, interleukins 1, 6, and 8. Systemic manifestations in human and experimental IBD can be explained by liberation of inflammatory mediators and toxic products from the inflamed intestine, which could produce lymphadenitis, splenitis, hepatitis, and arthritis (37). Systemic administration of IL-1, TNF, and IL-6 induces leukocytosis, normochromic anemia, and the acute-phase response (2, 37).

In this study, a most remarkable finding is an almost total inhibition (by 83% at days 17 and 18) of the increase in joint diameter by treatment with P8720. This compares to our previous study showing that P8720 attenuated (by 61% at 49 h) acute arthritis induced in Lewis rats by intraperitoneal PG-APS injection (24). Moreover, recent data indicate that endogenous kinins may mediate acute arthritis in this model, through B2 bradykinin receptors (38). However, arthritis associated with intestinal inflammation displays a progressive destructive course, with an onset at approximately 14 days after PG-APS injection, in contrast to the acute transient phase of arthritis induced by intraperitoneal PG-APS injection in Lewis rats, which peaks between days 3 and 5. Prevention of the increase of joint enlargement, white cell count, spleen and liver weight, and a decrease in hematocrit could be related in part to a decrease in monocyte/macrophage and neutrophil activation and to subsequent cytokine synthesis. This postulate is supported by an impressive decrease in the plasma T-kininogen level, an acute-phase reactant in which hepatic synthesis is stimulated by IL-6. Endogenous kinins are also involved in regulation of synthesis of this protein by the liver (39). Although treatment with P8720 inhibitor did not significantly modulate liver granuloma formation, kallikrein inhibition decreased hepatomegaly and significantly decreased intestinal granulomas.

Our studies in this model indicate that the K-K system is one determinant of genetic susceptibility to chronic intestinal and systemic inflammation. Activation of this pathway closely correlated with acute and chronic inflammation in Lewis rats, but did not occur in Buffalo rats even during acute inflammation, when Buffalo rats showed a greater degree of inflammation than Lewis rats (4). Furthermore, activation of the K-K system in Lewis rats correlated with the rate of cleavage of HK in vitro. Recent observations (A. Stadnicki, Y. Lin, R. B. Sartor, and R. W. Colman, unpublished data) suggests that an intrinsic difference in the activation of this system in rat strains is dependent on an HK polymorphism. The importance of kallikrein in the regulation of the acute phase of enterocolitis in this model can be deduced from the consequences of inhibition by P8720 (8). Finally, we showed that K-K system activation accompanies acute and chronic phases of enterocolitis in an entirely different model of intestinal inflammation in Lewis rats induced by indomethacin (10). Together, our data indicate that the K-K system is integrally involved in the pathogenesis of acute as well as chronic intestinal inflammation and systemic complications.

Morbidity in experimental IBD results from numerous pathogenic pathways, but in the present study we clearly demonstrate that specific inhibition of one, plasma kallikrein, very much alters the inflammatory response. Our previous study (24) has demonstrated that P8720 is at least 100-fold more potent on plasma kallikrein than on the coagulation enzymes (factor XIIa, factor Xa, thrombin), fibrinolytic enzymes (tissue plasminogen activator, plasmin), classic complement enzymes (C1r and C1s), and tissue kallikrein. We cannot rule out alternative effects of the inhibitor on other components involved in this model of intestinal inflammation, including effects on alternative and common pathways of complement or on T cells. In fact, the hepatic toxicity of P8720 at higher doses than those used in this study implies other pharmacologic effects. Nevertheless, our results provide evidence that a specific kallikrein inhibitor prevents chronic experimental intestinal and systemic inflammation, which yields insight into the pathogenesis of IBD and may have clinical therapeutic implications. Our data demonstrate activation of the K-K system in active ulcerative colitis patients (40). Unfortunately, high levels of P8720 elevate plasma transaminase in rats (8). In this study, we used a lower dose of P8720 because of documented hepatic toxicity with the dose used in our acute study (8). We observed a transaminase increase in only one of the eight rats treated with the lower dose, but did observe weight loss. This formulation may have such a narrow therapeutic range that it would be unacceptable for human use. However, nontoxic specific kallikrein inhibitors or, alternatively, bradykinin antagonists could be useful. The ideal treatment of the acute phase and the maintenance of remission in IBD still presents a major clinical challenge (41). Treatment and prevention of reactivation of IBD, due to the involvement of a number of pathogenic factors and mediators, probably will require combined therapy or novel approaches to block key immunoregulatory events such as activation or infiltration of TH1 lymphocytes.

	FOOTNOTES

 
¹ Correspondence: Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 N. Broad St., Philadelphia, PA 19140, USA. E-mail: colmanr{at}astro.temple.edu

² Abbreviations: IL-1, interleukin 1; IBD, inflammatory bowel diseases; K-K, kallikrein-kinin; FXII, zymogen factor XII (FXIIa, the active form); PK, prekallikrein; FXI, coagulation factor XI; GPT, glutamic pyruvic transaminase; HSA, human serum albumin; ATIII, antithrombin III; HK, high molecular weight kininogen; TNF, tumor necrosis factor; PG-APS, peptidoglycan-polysaccharide from group A streptococci.

Received for publication September 17, 1997. Accepted for publication November 12, 1997.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Podolsky, D. K. (1991) Inflammatory bowel disease (1). N. Engl. J. Med. 325, 928–937[Medline]
2. Sartor, R. B. (1995) Current concepts of the etiology and patho- genesis of ulcerative colitis and Crohn's disease. Gastroenterol. Clin. N. Am. 24, 475–508[Medline]
3. Elson, C. O., Sartor, R. B., Tennyson, G. S., and Riddell, R. H. (1995) Experimental models of inflammatory bowel disease. Gastroenterology 109, 1344–1367[Medline]
4. Sartor, R. B., DeLa Cadena, R. A., Green, K. D., Stadnicki, A., Davis, S. W., Schwab, J. H., Adam, A. A., Raymond, P., and Colman, R. W. (1996) Selective kallikrein-kinin system activation in inbred rats differentially susceptible to granulomatous enterocolitis. Gastroenterology 110, 1467–1481[Medline]
5. Schwab, J. H. (1993) Phlogistic properties of peptidoglycan-polysaccharide polymers from cell walls of pathogenic and normal-flora bacteria which colonize humans. Infect. Immun. 61, 4535–4539[Abstract/Free Full Text]
6. McCall, R. D., Haskill, S., Zimmermann, E. M., Lund, P. K., Thompson, R. C., and Sartor, R. B. (1994) Tissue interleukin 1 and interleukin-1 receptor antagonist expression in enterocolitis in resistant and susceptible rats. Gastroenterology 106, 960–972[Medline]
7. Sartor, R. B., Cromartie, W. J., Powell, D. W., and Schwab, J. H. (1985) Granulomatous enterocolitis induced in rats by purified bacterial cell wall fragments. Gastroenterology 89, 587–595[Medline]
8. Stadnicki, A., DeLa Cadena, R. A., Sartor, R. B., Bender, D., Kettner, C. A., Rath, H. C., Adam, A., and Colman, R. W. (1996) Selective plasma kallikrein inhibitor attenuates acute intestinal inflammation in Lewis rat. Dig. Dis. Sci. 41, 912–920[Medline]
9. Herforth, H. H., Mohanty, S. P., Rath, H. C., Tankonogy, S., and Sartor, R. B. (1996) Interleukin 10 suppresses experimental chronic, granulomatous inflammation induced by bacterial cell wall polymers. Gut 39, 836–845[Abstract/Free Full Text]
10. Stadnicki, A., Sartor, R. B., and Colman, R. W. (1996) Activation of the kallikrein-kinin system in indomethacin-induced enterocolitis in genetically susceptible rats. J. Invest. Med. 44, 299(Abstract)
11. Morrison, D. C., and Cochrane, C. G. (1974) Direct evidence for Hageman factor (factor XII) activation by bacterial lipopolysaccharides (endotoxins). J. Exp. Med. 140, 797–811[Abstract]
12. Kaplan, A. P., Kay, A. B., and Austen, K. F. (1972) A prealbumin activator of prekallikrein. III. Appearance of chemotactic activity for human neutrophils by the conversion of human prekallikrein to kallikrein. J. Exp. Med. 135, 81–97[Abstract]
13. Schapira, M., Despland, E., Scott, C. F., Boxer, L. A., and Colman, R. W. (1982) Purified human plasma kallikrein aggregates human blood neutrophils. J. Clin. Invest. 69, 1199–1202
14. Zimmerli, W., Huber, I., Bouma, B. N., and Lammle, B. (1989) Purified human plasma kallikrein does not stimulate but primes neutrophils for superoxide production. Thromb. Haemost. 62, 1121–1125[Medline]
15. Wachtfogel, Y. T., Kucich, U., James, H. L., Scott, C. F., Schapira, M., Zimmerman, M., Cohen, A. B., and Colman, R. W. (1983) Human plasma kallikrein releases neutrophil elastase during blood coagulation. J. Clin. Invest. 72, 1672–1677
16. Wachtfogel, Y. T., Pixley, R. A., Kucich, U., Abrams, W., Weinbaum, G., Schapira, M., and Colman, R. W. (1986) Purified plasma factor XIIa aggregates human neutrophils and causes degranulation. Blood 67, 1731–1737[Abstract/Free Full Text]
17. Toossi, Z., Sedor, J. R., Mettler, M. A., Everson, B., Young, T., and Ratnoff, O. D. (1992) Induction of expression of monocyte interleukin 1 by Hageman factor (factor XII). Proc. Natl. Acad. Sci. USA 89, 11969–11972[Abstract/Free Full Text]
18. Ghebrehiwet, B., Silverberg, M., and Kaplan, A. P. (1981) Activation of the classical pathway of complement by Hageman factor fragment. J. Exp. Med. 153, 665–676[Abstract/Free Full Text]
19. Ichinose, A., Fujikawa, K., and Suyama, T. (1986) The activation of prourokinase by plasma kallikrein and its inactivation by thrombin. J. Biol. Chem. 261, 3486–3489[Abstract/Free Full Text]
20. DiScipio, R. (1982) The activation of the alternative pathway C3 convertase by human plasma kallikrein. Immunology. 45, 587–595[Medline]
21. DeLa Cadena, R. A., Wachtfogel, Y. T., and Colman, R. W. (1994) Contact activation pathway: Inflammation and coagulation. In Hemostasis and Thrombosis: Basic Principles and Clinical Practice (Colman, R. W., Hirsh, J., Marder, V. J., and Salzman, E. W., eds) pp. 219–240, J. B. Lippincott, Philadelphia
22. Bhoola, K. D., Figueroa, C. D., and Worthy, K. (1992) Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmacol. Rev. 44, 1–80[Medline]
23. Gaginella, T. S., and Kachur, J. F. (1989) Kinins as mediators of intestinal secretion. Am. J. Physiol. 256, G1–15[Abstract/Free Full Text]
24. DeLa Cadena, R. A., Stadnicki, A., Uknis, A. B., Sartor, R. B., Kettner, C. A., Adam, A., and Colman, R. W. (1995) Inhibition of plasma kallikrein prevents peptidoglycan-induced arthritis in the Lewis rat. FASEB J 9, 446–452[Abstract/Free Full Text]
25. DeLa Cadena, R. A., Scott, C. F., and Colman, R. W. (1987) Evaluation of a microassay for human plasma prekallikrein. J. Lab. Clin. Med. 109, 601–607[Medline]
26. Scott, C. F., and Colman, R. W. (1988) A simple and accurate microplate assay for the determination of factor XI in plasma. J. Lab. Clin. Med. 111, 708–714[Medline]
27. Proctor, R. R., and Rapaport, S. I. (1961) The partial thromboplastin time with kaolin: A simple screening test for first stage plasma clotting factor deficiencies. Am. J. Clin. Pathol. 36, 212–219[Medline]
28. Colman, R. W., Bagdasarian, A., Talamo, R. C., Scott, C. F., Seavey, M., Guimaraes, J. A., Pierce, J. V., and Kaplan, A. P. (1975) Williams trait. Human kininogen deficiency with diminished levels of plasminogen proactivator and prekallikrein associated with abnormalities of the Hageman factor-dependent pathways. J. Clin. Invest. 56, 1650–1662
29. Scott, C. F. (1985) Determination of antithrombin III (AT-III) using the Coatest Antithrombin Kit and a microplate system. Ann. N.Y. Acad. Sci. 485, 443–440
30. Adam, A., Damas, J., Calay, G., Renard, C., Remacle Volon, G., and Bourdon, V. (1989) Quantification of rat T-kininogen using immunological methods. Application to inflammatory processes. Biochem. Pharmacol. 38, 1569–1575[Medline]
31. Bergmeyer, H. U., Scheibe, P., and Wahlefeld, A. W. (1978) Optimization of methods for aspartate aminotransferase and alanine aminotransferase. Clin. Chem. 24, 58–73[Abstract/Free Full Text]
32. Murphy, E. A. (1982) Biostatistics in Medicine, The Johns Hopkins Press, Baltimore
33. Pixley, R. A., DeLa Cadena, R. A., Page, J. D., Kaufman, N., Wyshock, E. G., Chang, A., Taylor, F. B., Jr., and Colman, R. W. (1993) The contact system contributes to hypotension but not disseminated intravascular coagulation in lethal bacteremia: In vivo use of a monoclonal anti-factor XII antibody to block contact activation in baboons. J. Clin. Invest. 91, 61–68
34. Sartor, R. B., Bender, D. E., Allen, J. B., Zimmerman, E. M., Holt, L. C., Pardo, M. S., Lund, P. K., and Wahl, S. M. (1993) Chronic experimental enterocolitis and extraintestinal inflammation are T lymphocyte dependent. Gastroenterology 104, 808A (abstr.)
35. Woolverton, C. J., White, J. J., Jr., and Sartor, R. B. (1989) Eicosanoid regulation of acute intestinal vascular permeability induced by intravenous peptidoglycan-polysaccharide polymers. Agents Actions 26, 301–309[Medline]
36. Jansen, P. M., Pixley, R. A., Brouwer, M., DeJong, I. W., Chang, A. K., Hack, C. E., Taylor, F. B., Jr., and Colman, R. W. (1996) Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase. Blood 87, 2337–2344[Abstract/Free Full Text]
37. Sartor, R. B., and Lichtman, S. N. (1994) Mechanisms of systemic inflammation associated with intestinal injury. In Inflammatory Bowel Disease: From Bench to Bedside (Targan, S. R., and Shanahan, F., eds) pp. 210–229, Williams & Wilkins, Philadelphia
38. Blais, C., Jr., Couture, R., Drapeau, G., Colman, R. W., and Adam, A. A. (1997) Endogenous kinins are involved in the pathogenesis of peptidoglycan-induced arthritis in the Lewis rat. Arthritis Rheum. 40, 1327–1333[Medline]
39. Raymond, P., Bouvier, M., Drapeau, G., Blais, C., Morais, R., and Adam, A. (1996) Bradykinin decreases T-kininogen synthesis in a rat hepatoma cell line: Evidence of bradykinin B2-type receptors. Peptides 17, 1171–1176[Medline]
40. Stadnicki, A., Gonciarz, M., Niewiarowski, T., Hartleb, J., Rudnicki, M., Merrel, N. B., DeLa Cadena, R. A., and Colman, R. W. (1997) Activation of plasma contact and coagulation systems and neutrophils in the active phase of ulcerative colitis. Dig. Dis. Sci. 42, 2356–2366[Medline]
41. Hanauer, S. (1995) Inflammatory bowel disease. N. Engl. J. Med. 331, 841–848