Secretory leukocyte protease inhibitor suppresses the inflammation and joint damage of bacterial cell wall-induced arthritis

Abstract

Disruption of the balance between proteases and protease inhibitors is often associated with pathologic tissue destruction. To explore the therapeutic potential of secretory leukocyte protease inhibitor (SLPI) in erosive joint diseases, we cloned, sequenced, and expressed active rat SLPI, which shares the protease-reactive site found in human SLPI. In a rat streptococcal cell wall (SCW)-induced model of inflammatory erosive polyarthritis, endogenous SLPI was unexpectedly upregulated at both mRNA and protein levels in inflamed joint tissues. Systemic delivery of purified recombinant rat SLPI inhibited joint inflammation and cartilage and bone destruction. Inflammatory pathways as reflected by circulating tumor necrosis factor alpha and nuclear factor kappaB activation and cartilage resorption detected by circulating levels of type II collagen collagenase-generated cleavage products were all diminished by SLPI treatment in acute and chronic arthritis, indicating that the action of SLPI may extend beyond inhibition of serine proteases.

Figure 1

cDNA sequence of rSLPI and amino acid sequence comparison among human, murine, and rat SLPI. (A) Nucleotide sequence of rSLPI is shown in plainface, and the amino acid sequence of the mature protein in boldface below the nucleotide sequence. The amino acid sequence for the signal peptide is underlined. Asterisk, stop codon. (B) Comparison of amino acid sequences of human, murine, and rat SLPI using ClustalW alignment. Conserved residues are shaded in dark color, and conserved cysteine residues are indicated by asterisks. The active site for protease binding is underlined with the two key residues double underlined.

Figure 5

(A) rrSLPI inhibits TNF-α production. Plasma samples of SCW-injected (hatched bar) and SCW animals that have received 0.1 mg of active rrSLPI on days 1 and 9 (solid bar) were collected as indicated and analyzed by ELISA. Each point represents the mean ± SEM (n = 6). **P < 0.01. (B) rrSLPI suppresses NF-κB activity in SCW animals. Proteins were extracted from joint samples of PBS-injected nonarthritic and SCW-injected and active rrSLPI-treated SCW arthritic animals 3 d after SCW or PBS and 2 d after rrSLPI injection and subjected to EMSA.

Figure 5

(A) rrSLPI inhibits TNF-α production. Plasma samples of SCW-injected (hatched bar) and SCW animals that have received 0.1 mg of active rrSLPI on days 1 and 9 (solid bar) were collected as indicated and analyzed by ELISA. Each point represents the mean ± SEM (n = 6). **P < 0.01. (B) rrSLPI suppresses NF-κB activity in SCW animals. Proteins were extracted from joint samples of PBS-injected nonarthritic and SCW-injected and active rrSLPI-treated SCW arthritic animals 3 d after SCW or PBS and 2 d after rrSLPI injection and subjected to EMSA.

Figure 2

SLPI expression in SCW-induced arthritis. (A) RNAs from PMNs, PBMCs, and PMs of control and SCW arthritic (day 26) animals were probed with rSLPI and rat GAPDH cDNA probes. (B) Total RNA from ankle tissues at indicated times after SCW injection were blotted to a Nytran membrane and probed with rSLPI cDNA. The ethidium bromide–stained gel demonstrates the amount of 28S and 18S RNAs. Data are representative of three experiments. (C–F) Immunohistochemical detection of rSLPI protein (brown stain) in joint sections before (C), 4 d after (D), and 26 d after SCW injection (E, F). Rabbit IgG was used as control in F. Syn, synovium; JS, joint space; C, cartilage; BM, bone marrow.

Figure 3

Expression and purification of active rrSLPI. (A) Diagram of HIS–rSLPI fusion protein for step-elution from Ni-NTA column. (B) SDS-PAGE of purified proteins: 1, sonicated induced cells; 2, lysate from 15,000 g centrifugation; 3, dialyzed lysate from the sequential 50,000 g centrifugation; 4, 75 mM imidazole elute fractions; MW, markers. (C) Immunoblot of B using anti-rSLPI peptide antibody as described in Materials and Methods. (D) rrSLPI (lane 4) possesses antiprotease activity toward human neutrophil elastase (shown), cathepsin G, and chymotrypsin. Different concentrations of SLPI were shown on the x-axis, and the fractional elastase activity at each inhibitor concentration compared with uninhibited activity was shown on the y-axis.

Figure 4

Suppression of arthritis by rrSLPI. (A) Arthritis was initiated by a single intraperitoneal injection of SCW on day 0 and the articular index (AI) measured as described in Materials and Methods for the course of the study (○). An intraperitoneal injection of 0.1 mg of the purified active rrSLPI protein was administered 24 h and again on day 9 after SCW (•) or only on day 13 (□). Each point represents the mean AI ± SEM for each group of animals (n = 12) and significant decreases in AI with treatment are indicated as *P < 0.05 and **P < 0.01. (B and C) Radiographs of representative animals 26 d after SCW injection (B) and rrSLPI-treatment on days 1 and 9 (0.1 mg) (C). (D) The effect of truncated rrSLPI (0.1 mg, hatched bars) administered on days 1 and 9 after SCW on acute and chronic arthritis was compared with that of the full-length rrSLPI (solid bars). The articular index of untreated SCW-injected animals was shown as open bars. Each point represents the mean AI ± SEM for each group of animals (n = 12) and significant differences are indicated by *P < 0.05 and **P < 0.01. (E) rrSLPI suppresses the increased cleavage of type II collagen. Plasma samples were collected during the acute (day 4), remission (day 10), and chronic phases (day 26) of arthritis and Col2-3/4C long neoepitope quantified by ELISA. Data shown are the means ± SEM for each group of animals (n = 6). SCW+rrSLPI represents the group of animals that have been injected with active rrSLPI on day 1 and 9 after SCW (solid bars) and PBS represents the control animals injected with phosphate buffered saline (open bars). *P < 0.05, **P < 0.001.