Borrelia spirochetes upregulate release and activation of matrix metalloproteinase gelatinase B (MMP-9) and collagenase 1 (MMP-1) in human cells

Abstract

Borrelia burgdorferi, the spirochetal agent of Lyme disease, stimulated human peripheral blood monocytes to release pro-matrix metalloproteinase-9 (gelatinase B; pro-MMP-9) and active matrix metalloproteinase-1 (collagenase-1; MMP-1). Human neutrophils also released pro-MMP-9 and a 130-kDa protein with gelatinolytic activity in response to live B. burgdorferi. In addition, U937 cells and human keratinocyte cells were also stimulated to release pro-MMP-9 under the same conditions. However, human umbilical vein endothelial cells (HUVECs) released pro-MMP-9 and pro-MMP-2 in a constitutive manner and were not influenced by live spirochetes. MMPs produced by human monocytes also enhanced the penetration of B. burgdorferi through extracellular matrix component barriers in vitro. Plasmin stabilized on the surface of the Lyme disease spirochete was shown to activate pro-MMP-9 to its active form. This active form was also observed in the plasma of mice infected with a relapsing fever borrelia. These results suggest that borreliae can upregulate MMPs and possibly mediate an activation cascade initiated by plasmin bound to the microbial surface. MMPs may play a role in dissemination of the Lyme disease spirochete and in the pathogenesis of Borrelia infection.

FIG. 1

Gelatin zymogram of pro-MMP-9 release into culture supernatant in response to B. burgdorferi by U937 cells. (a) U937 cells were cultured with either 0 (no spirochetes), 1, 10, 100, or 500 B. burgdorferi (strain N40) per cell. (b) Increasing levels of mmp-9 mRNA were detected in response to 0, 10, and 100 B. burgdorferi per U937 cell by RT-PCR using mmp-9-specific primers. PMA was used a positive control. Levels of β-actin mRNA were not influenced by increasing numbers of B. burgdorferi.

FIG. 2

B. burgdorferi upregulates release of MMP-9 and MMP-1 from human peripheral blood monocytes. (a) Gelatin zymography of human peripheral blood monocytes, showing increasing levels of pro-MMP-9 released following coculture with 0 (no spirochetes), 10, 50, or 100 B. burgdorferi per cell. Sh., sham-infected control. (b) Western blot analysis showing that the gelatinolytic activity observed at 92 kDa in panel is pro-MMP-9. (c) Western blot of pro- and active MMP-1 from human peripheral blood monocytes following coculture with 0 (no spirochetes), 10, 50, or 100 B. burgdorferi per cell. Pos., positive control (bovine serum). Top arrow, latent forms of MMP-1 (57 or 52 kDa); bottom arrow, active forms (46 or 42 kDa).

FIG. 3

B. burgdorferi upregulates the release of pro-MMP-9 in human keratinocytes but not endothelial cells. (a) Gelatin zymography of culture supernatant from primary human keratinocytes following coculture with 50 B. burgdorferi per cell and with 100 ng of LPS, showing release of pro-MMP-9. Control received no spirochetes or LPS. (b) Gelatin zymography of culture supernatant from primary human endothelial cells following coculture with 0 (no spirochetes), 10, 50, 100, and 500 B. burgdorferi, showing constitutive release of both pro-MMP-9 and pro-MMP-2. Sh., sham-infected control.

FIG. 4

Human neutrophils upregulate the release of pro-MMP-9 and cross-reactive complexes in response to B. burgdorferi. (a) SDS-PAGE of culture supernatant of human neutrophils cocultured with 8 live B. burgdorferi per neutrophil. Lane 1, no spirochetes; lane 2, with spirochetes; lane 3, molecular size standards. (b) Western blot of culture supernatant from human neutrophils following coculture with 0, 0.3, 3.0, or 8.0 B. burgdorferi per cell for either 30 or 60 min, showing the presence of pro-MMP-9 and a cross-reactive 130-kDa protein.

FIG. 5

Plasminogen and urokinase bound to spirochetes activate MMPs. (a) Gelatin zymography of culture supernatant from human peripheral blood monocytes cocultured with 10, 50, and 100 B. burgdorferi per cell with or without bound plasmin. Borreliae with bound plasmin resulted in elevated levels of active MMP-9 (arrow). (b) Gelatin zymography of culture supernatant from U937 cells cocultured with 100 and 500 B. burgdorferi per cell with or without bound plasmin. Borreliae with bound plasmin resulted in elevated levels of active MMP-9. (c) Mice infected with a relapsing fever borrelia exhibited elevated plasma levels of active MMP-9 (arrow) at peak spirochetemia.

FIG. 6

Human peripheral blood monocytes enhance penetration of B. burgdorferi (Bb) across extracellular matrix components. Monocytes (PBMs) enhanced penetration of B. burgdorferi at 24 h across (a) collagen type I, (b) laminin, and (c) collagen type IV. Inhibition of penetration was observed at 24 h across (a) collagen type I, (b) laminin, and (c) collagen type IV using an MMP inhibitor (inhib.).