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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Objective

To compare inflammatory peripheral arthritis in wild-type and high molecular weight kininogen (HK)–deficient rats, both on the genetically susceptible Lewis background.

Methods

By backcrossing Brown-Norway HK-deficient rats with Lewis rats for 6 generations, 2 new strains were produced, wild-type F6 and HK-deficient F6, each with a 98.5% Lewis genome. Inflammatory arthritis was induced by intraperitoneal injection of peptidoglycan–polysaccharide (PG-PS), and the clinical, histopathologic, and biochemical responses were compared in both strains.

Results

Eighteen days after PG-PS injection, rats with normal concentrations of HK showed weight loss and marked increase in hind ankle diameter with severe synovial inflammation and cartilage abnormalities. In contrast, HK-deficient rats showed no weight loss (P < 0.05), no increase in hind ankle diameter (P < 0.05), and an absence of inflammatory changes (P < 0.05), as measured by the histologic and morphometric Mankin grading system for synovial and cartilage injury.

Conclusion

Plasma HK is a key mediator of acute and chronic inflammatory arthritis in genetically susceptible Lewis rats.

Intraperitoneal administration of peptidoglycan–polysaccharide (PG-PS) induces inflammatory changes in the hind ankles of inbred female Lewis rats within 72 hours, which resolve in a few days. These changes are followed by chronic T cell–mediated erosive arthritis that persists for several weeks with relapses and partial remissions (1). This inflammatory response is accompanied by activation of the plasma kallikrein-kinin system (KKS), which leads to generation of biologically active effector molecules. The KKS in humans and rats consists of 4 major proteins: factor XII (FXII), factor XI (FXI), prekallikrein (PK), and high molecular weight kininogen (HK). Activated FXII (FXIIa) stimulates neutrophil aggregation (2) and up-regulates interleukin-1 (IL-1) expression in monocytes (3). HK complexed with PK binds to the endothelial surface, where Hsp90 (4), prolylcarboxypeptidase (5), or FXIIa cleaves the HK–PK complex to HK–kallikrein. This process is further amplified on endothelial cells by kallikrein cleavage of FXII to FXIIa.

Kallikrein in the presence of HK stimulates mononuclear chemotaxis (6) and induces the release of neutrophil elastase. Plasma kallikrein cleaves human and rat HK, which releases bradykinin (BK). BK stimulates intestinal inflammation, releases prostaglandins and nitric oxide, and enhances microvascular flow and permeability (7). The resulting active cofactor, HKa, undergoes dramatic conformational changes and binds to anionic-charged surfaces and endothelial cell receptors (3). Preliminary studies showed that HKa induces tumor necrosis factor (TNF) secretion in rats and IL-1β secretion in humans (through the urokinase-type plasminogen activator receptor and CD11/CD18 complex [Mac-1]) (8). To investigate the role of HK in reactive arthritis, we created kininogen-deficient Lewis rats by backcrossing the Brown-Norway rat Katholiek strain (HK-deficient) with normal Lewis rats (9) and compared the ability of intraperitoneal PG-PS to induce arthritis in both.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Production of HK-deficient Lewis rats

Lewis rats have a mutation, S511N, in kininogen (10) that results in accelerated cleavage of HK and increased susceptibility to chronic inflammatory processes. The Brown-Norway rat Katholiek strain has a mutation in HK, A143T, characterized by a hepatic secretion defect that results in plasma HK levels ranging from 5% to 10% of normal (2). Since the Brown-Norway wild-type strain is resistant to PG-PS–induced arthritis, we backcrossed the heterozygous offspring of a Brown-Norway Katholiek rat and a Lewis rat that mated with Lewis rats for 6 generations. After 6 backcrosses, the Lewis genome represented 98.5% of the total gene pool. In the fifth generation, the heterozygous rats were crossed and homozygous HK-deficient and wild-type HK founders were identified. Breeding the homozygous HK-deficient founders and the normal HK rats with each other produced 2 new strains: HK-deficient (F6HKd) and wild-type F6 (F6WT; with normal HK plasma levels).

PG-PS polymers

Purified, sterile PG-PS fragments from cell walls of group A, type 3, strain D58 streptococci (Streptococcus pyogenes) were prepared as previously described (1).

Animal protocol

All F6 Lewis rats, HK-deficient (n = 8; mean ± SEM weight 303.30 ± 14.34 gm) and wild-type (n = 5; 352.00 ± 20.35 gm), were bred at the University of North Carolina, Department of Laboratory Animal Medicine, Rodent Housing Facility, and then were transferred to and housed in the Temple University Weiss Hall Animal Care Facility, with free access to food and water. The protocol was approved by the Institutional Animal Care and Use Committee before implementation and conformed to the National Institutes of Health guidelines. Each animal received a single intraperitoneal injection of 15 μg PG-PS (rhamnose equivalent)/gm of body weight on day 0 and was followed up for 18 days. Total body weight and hind paw diameters were recorded daily from day 0 to day 6 and every other day afterward until day 18. The ankle joint diameter of each hind paw was measured in triplicate with a digital caliper (Ultra-Cal Mark III; FV Fowler, Newton, MA). On day 18, all animals were anesthetized and killed by exsanguination (cardiac puncture). Liver, spleen, and hind paws were removed and fixed in 10% buffered formalin (Fisher Scientific, Fair Lawn, NJ).

Plasma KKS functional assays

Blood samples were obtained by left atrium puncture as the heart beat, with slow vacuum (to prevent hemolysis) within 1 minute (to prevent clotting), transferred, and gently mixed in Eppendorf polypropylene tubes (Fisher Scientific) containing 100 μl of anticoagulant (citrate–phosphate–dextrose solution with adenine; Sigma, St. Louis, MO) to a final volume of 1 ml. Plasma was isolated by double centrifugation (3,000 revolutions per minute for 10 minutes and 10,000 rpm for 10 minutes at 23°C) in polypropylene tubes; supernatant aliquots were stored at −70°C. PK function levels were determined using a microtiter, amidolytic assay. FXI, FXII, and HK were measured using specific deficient plasma. All assays were performed in our laboratories at Temple University, as previously described (9). One milliliter of pooled F6WT rat plasma was defined as containing 1 unit/ml each of rat HK, PK, FXI, and FXII.

Histopathology

The liver and spleen were weighed after fixation, paws were decalcified in formic acid (Fisher Scientific), and all specimens were submitted for paraffin embedding, sectioning, and hematoxylin and eosin staining. The paws were also stained with Safranin O–fast green (Fisher Scientific) for cartilage proteoglycan content evaluation. The degree of inflammation and joint damage was measured using the Mankin grading system (11). Briefly, synovial tissue from tarsal joints was examined for synovial hyperplasia, fibrosis, inflammatory cell infiltrates, and pannus formation. The total histologic score for synovial inflammation ranged from 0 to 11. Articular cartilage histologic evaluation included cartilage organization changes, chondrocyte proliferation, proteoglycan content, and tidemark integrity. The total cartilage Mankin score ranged from 0 to 14.

Statistical analysis

Data are expressed as the mean ± SEM. All parameters were subjected to Student's unpaired t-test. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Total body weight. Both groups showed a loss in total body weight by day 2 (F6HKd group mean ± SEM −24.8 ± 4.7 gm and F6WT group −49.0 ± 14.8 gm). The F6WT group lost weight from days 7 to 16 and had a slight improvement on day 18 (−44.40 ± 7.30 gm). The F6HKd group slowly gained weight from days 6 to 18 (−13.25 ± 5.85 gm). The initial weight loss of the F6WT group was more prominent than that of the F6HKd group and mirrored the severity of the arthritis during the length of the study (P < 0.05).

Changes in joint diameter. The F6HKd group showed no signs of arthritis (joint diameter increase) during the study (Figure 1). The mean ± SEM hind ankle diameter at baseline was 5.83 ± 0.036 cm in the F6HKd group and 5.80 ± 0.035 cm in the F6WT group. The F6WT group developed a biphasic increase in joint diameter. The acute phase started on day 1 (increase of 0.13 ± 0.05 cm) and peaked on day 5 (increase of 0.25 ± 0.04 cm), at which point it reached a plateau, merged into the chronic phase that started on day 7 (increase of 0.28 ± 0.05 cm), and continued through day 18 (increase of 1.10 ± 0.5 cm).

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Figure 1. Joint diameter changes. F6HKd rats, which were deficient in high molecular weight kininogen (n = 8) (solid circles), developed no signs of arthritis. Normal F6WT rats (n = 5) (open circles) showed a steady increase in the diameter of the hind ankle joint throughout the study. Values are the mean and SEM. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.005. PG-PS = peptidoglycan–polysaccharide.

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Hind paw histologic assessment. The F6HKd group showed no inflammatory changes. The F6WT group exhibited diffuse inflammatory infiltration with severe synovial thickening, pannus formation, cartilage destruction with loss of proteoglycan content, and bone erosion (Figure 2). The mean ± SEM synovial inflammation score was 3.90 ± 1.04 in the F6WT group and 0.19 ± 0.09 in the F6HKd group (P = 0.0008), and the cartilage score was 5.00 ± 1.21 in the F6WT group and 0.06 ± 0.06 in the F6WT group (P = 0.0003). The total Mankin score was 4.5 ± 0.08 and 0.13 ± 0.06 in the F6WT group and the F6HKd group, respectively (P < 0.001), with significant differences (P < 0.05) in both synovitis and cartilage damage components.

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Figure 2. Histologic findings in the hind ankles of F6HKd rats, which were deficient in high molecular weight kininogen, and normal F6HKWT rats. A and B, Photomicrographs of specimens from F6HKd rats, showing normal joint space (a) and bone (b). Arrow in B shows normal cartilage peptidoglycan contents (red). C and D, Photomicrographs of specimens from F6HKWT rats. Arthritis is evident, with pannus (c) occupying the joint space and infiltrating bone. Arrow in D shows decreased peptidoglycan contents. (Hematoxylin and eosin stained in A and C, Safranin O–fast green stained in B and D; original magnification × 100.)

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Organ histology assessment. The F6HKd group exhibited liver granulomas in 1 of 8 animals and spleen granulomas in 6 of 8. In the F6WT group, liver granulomas were found in 2 of 5 animals and spleen granulomas in 5 of 5. These differences were not significant.

KKS functional assays. Injection of PG-PS caused a significant decrease (P < 0.05) in the concentration of HK (mean ± SEM 0.59 ± 0.09 units/ml), PK (0.40 ± 0.05 units/ml), and FXI (0.80 ± 0.05 units/ml) in F6WT rats when compared with pooled plasma from F6WT rats, which indicated in vivo activation of KKS secondary to PG-PS challenge (Figure 3). No significant differences were found in FXII levels. Uninjected pooled F6HKd rat plasma had lower levels of HK (0.08 ± 0.0001 units/ml) and PK (0.54 ± 0.004 units/ml) than pooled F6WT rat plasma, but the levels of FXI and FXII were similar. When F6HKd rats were challenged with PG-PS injection, their levels of HK, FXI, and FXII were unchanged, while the concentration of PK was significantly decreased (0.32 ± 0.05 units/ml; P = 0.043).

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Figure 3. Functional protein levels of the kallikrein-kinin system (KKS) in normal F6WT rats (n = 3) (crosshatched bars), peptidoglycan–polysaccharide (PG-PS)–injected F6WT rats (n = 4) (open bars), F6HKd rats, which were deficient in high molecular weight kininogen (n = 4) (hatched bars), and F6HKd PG-PS–injected rats (n = 8) (solid bars). The significance of the difference in levels of content factors in the 2 F6 strains was compared versus their respective pools. Values are the mean and SEM. ∗ = P < 0.05; ∗∗∗ = P < 0.005. PK = prekallikrein; XI = factor XI; XII = factor XII.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The PG-PS–induced inflammatory response includes biphasic chronic granulomatous intestinal inflammation (intramural injection) and inflammatory arthritis (intraperitoneal injection). Activation of KKS occurs with a decrease of its component plasma proteins in experimental inflammatory bowel disease (IBD) and arthritis; significant disease modulation follows administration of a kallikrein inhibitor in IBD and arthritis, and administration of C11C1, a monoclonal antibody targeting HK, in arthritis (12). Therapeutic effects of C11C1 on colitis and arthritis in HLA–B27–transgenic rats have also been described (13). We created kininogen-deficient rats on a susceptible Lewis background strain and found that this group failed to develop PG-PS–induced arthritis. Thus, although KKS activation is not the only mechanism in this model of inflammatory arthritis, HK deficiency ameliorates the development of systemic inflammatory response secondary to PG-PS. Similar responses with pharmacologic inhibition of kallikrein, antibody blockade, and genetic deficiency of kininogen firmly implicate KKS in the pathogenesis of systemic inflammation in this experimental model.

We found that the plasma PK concentration was low in F6HKd rats, similar to findings in human HK deficiencies. This phenomenon is due to the fact that PK circulates bound to HK and has increased reactivity in the functional assay (2). In HK-deficient human plasma, the addition of purified HK normalizes the PK activity.

Cleavage of HK yields products that mediate inflammatory events such as leukocyte recruitment (14) and neovascularization in inflammatory states and in tumor growth (15). HK effects on angiogenesis are most likely mediated by BK, which can induce vascular endothelial growth factor expression by endothelial cells (16). Our preliminary studies demonstrate that one of the cleavage products, HKa, can induce monocyte production of IL-1β and TNF. These inflammatory cytokines have been implicated in the pathogenesis of experimental PG-PS–induced arthritis and enterocolitis in Lewis rats (2). HK deficiency has been shown to inhibit the development of angiogenesis (15) and block the production of inflammatory exudates (2, 14).

The present study shows that HK deficiency modulates the acute and chronic phases of inflammatory arthritis, demonstrating that plasma HK and its cleavage products are key mediators of inflammation in this rat model. Our results support the hypothesis that therapeutic approaches targeting the HK molecule have considerable potential in a varied range of human inflammatory disease states.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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