Potential involvement of gelatinases and their inhibitors in Mannheimia haemolytica pneumonia in cattle

Abstract

Mannheimia haemolytica infection of the lower respiratory tract of cattle results in a bronchofibrinous pneumonia characterized by massive cellular influx and lung tissue remodeling and scarring. Since altered levels of gelatinases and their inhibitors have been detected in a variety of inflammatory conditions and are associated with tissue remodeling, we examined the presence of gelatinases in lesional and nonlesional lung tissue obtained from calves experimentally infected with M. haemolytica. Lesional tissue had elevated levels of progelatinase A and B and active gelatinase A and B when compared with nonlesional tissue obtained from the same lung lobe. In vitro, M. haemolytica products stimulated production of gelatinase B, but not its activation, by bovine monocytes. Alveolar macrophages showed constitutive production of gelatinase B but no change in response to M. haemolytica products. Bovine neutrophils exposed to M. haemolytica products also released gelatinase B, and there was a significant increase in the activated form of this enzyme. These effects were virtually identical when recombinant O-sialoglycoprotease was used to stimulate these cells. M. haemolytica products also enhanced the expression by bovine monocytes and alveolar macrophages of the tissue inhibitor of metalloproteinase 1. Our results provide evidence that matrix metalloproteinases are activated in lung lesions from cattle with shipping fever and that M. haemolytica virulence products induce production, release, and especially activation of gelatinase B by bovine inflammatory cells in vitro.

FIG. 1.

(A) Zymography showing gelatinase activity in tissue lysates from nonlesional (N) and lesional (L) lung tissue obtained from calves 6 days after challenge with M. haemolytica. Lysis bands representing both progelatinase A and B and activated gelatinase A (MMP-2) and B (MMP-9) are more prominent in samples from lesion areas of lung. (B) Densitometric analysis of zymography from lesion and nonlesion areas of lungs from six animals. Open bar, mean density (± SEM) of gelatinases in nonlesional tissue; filled bar, mean density (± SEM) of gelatinases in lesional tissue; *, significantly higher levels compared to nonlesional tissue for each enzyme (P < 0.05); †, significantly higher levels of progelatinase B than active gelatinase B in nonlesional tissue (P < 0.05); ††, significantly higher levels of progelatinase A than active gelatinase A in lesional tissue (P < 0.05). Gel, gelatinase; Pro Gel, progelatinase.

FIG. 2.

(A) Representative zymography of gelatinase B activity in monocytes stimulated with M. haemolytica products. M, M. haemolytica supernatant; H, HTCS; C, control (vehicle only) conditions. (B) Representative zymography of gelatinase B activity in monocytes stimulated with the following: G, M. haemolytica recombinant Gcp; H, HTGcp; C, control conditions. (C) Densitometric analysis of zymography gels from four separate experiments, showing the mean density (± SEM) of gelatinase B from monocytes stimulated with CS (black), HTCS (gray), or control (vehicle only) conditions (white). (D) Densitometric analysis of zymography gels from four separate experiments, showing the mean density (± SEM) of gelatinase B from monocytes stimulated with recombinant Gcp (black), HTGcp (gray), or under control conditions (white). *, significant difference from control at the same time point (P < 0.05).

FIG. 3.

(A) Representative zymography of gelatinase B activity in bovine neutrophil releasates from cells stimulated with M. haemolytica CS, HTCS, Gcp, HTGcp, or control conditions (vehicle only) for 30 min in vitro. Bands of approximately 82 kDa, indicative of activated gelatinase B, were prominent in releasates from cells stimulated with active but not heat-treated M. hemolytic products. (B) Densitometric analysis of zymography gels from six separate experiments, showing the mean density (± SEM) of gelatinase B from cells stimulated under control conditions (white bars) or with HTCS (gray bars) or CS (black bars). Stimulation with M. haemolytica products induced extensive release of gelatinase B in relation to the level released under control conditions. (C) Densitometric analysis of zymography gels from four separate experiments, showing the mean density (± SEM) of gelatinase B from cells stimulated with control (white bars), HTGcp (gray bars), or Gcp (black bars). *, significantly higher levels of progelatinase B than under control conditions (P < 0.05); **, significantly higher levels of active gelatinase B than for control or under heat-treated conditions (P < 0.05). Gel, gelatinase; Pro Gel, progelatinase.

FIG. 4.

(A) Composite reverse zymography of TIMP activity in monocytes stimulated with M. haemolytica products. M, M. haemolytica supernatant; H, HTCS; C, control (vehicle) conditions. Purified mouse TIMPs (T) were run as standards. (B) Composite reverse zymography of TIMP activity in monocytes stimulated with recombinant M. haemolytica Gcp (G) or HTGcp (H) or under control (vehicle) conditions (C). Purified mouse TIMPs (T) were run as standards. (C) Densitometric analysis of reverse zymography gels from four separate experiments. Columns represent the mean density (± SEM) of TIMP-1 from monocytes stimulated with CS (black), HTCS (gray), or under control conditions (white). (D) Densitometric analysis of reverse zymography gels from four separate experiments. Columns represent the mean density (± SEM) of TIMP-1 from monocytes stimulated with Gcp (black), HTGcp (gray), or under control conditions (white). *, significant difference from control at the same time point; †, significant difference from heat-treated counterpart at the same time point (P < 0.05).

FIG. 5.

(A) Representative reverse zymography of TIMP activity in alveolar macrophages stimulated with M. haemolytica products for 4 h. M, M. haemolytica supernatant; H, HTCS; C, control conditions. Purified mouse TIMPs (T) were run on the gel as standards. (B) Densitometric analysis of reverse zymography gels from four separate experiments. Columns represent the mean density (± SEM) of TIMP-1 from alveolar macrophages stimulated with CS (black), HTCS (gray), or under control conditions (white). *, significant difference from control and heat-treated sample at the same time point (P < 0.05).