Localization of plasmin on macrophages and activation of pro-MMP-9 play key roles in macrophage recruitment in the inflammatory response. These functions are promoted by plasminogen receptors exposing C-terminal basic residues on the macrophage surface. Recently, we identified a novel transmembrane plasminogen receptor, Plg-R KT , which exposes a C-terminal lysine on the cell surface. In the present study, we investigated the role of Plg-R KT in macrophage invasion, chemotactic migration, and recruitment. Plg-R KT was prominently expressed in membranes of human peripheral blood monocytes and monocytoid cells. Plasminogen activation by urokinasetype plasminogen activator (uPA) was markedly inhibited (by 39%) by treatment with anti-Plg-R KT mAb. Treatment of monocytes with anti-Plg-R KT mAb substantially inhibited invasion through the representative matrix, Matrigel, in response to MCP-1 (by 54% compared with isotype control). Furthermore, chemotactic migration was also inhibited by treatment with anti-Plg-R KT mAb (by 64%). In a mouse model of thioglycollate-induced peritonitis, anti-Plg-R KT mAb markedly inhibited macrophage recruitment (by 58%), concomitant with a reduction in pro-MMP-9 activation in the inflamed peritoneum. Treatment with anti-Plg-R KT mAb did not further reduce the low level of macrophage recruitment in plasminogennull mice. We conclude that Plg-R KT IntroductionActivation of plasminogen, the zymogen of the primary thrombolytic enzyme plasmin, is markedly promoted when plasminogen is bound to cell surfaces (for review, see Miles et al 1 ) and cellassociated plasmin is protected from inactivation. 2,3 Therefore, cells become armed with the broad-spectrum proteolytic activity of plasmin. 4 This provides a mechanism to facilitate both physiologic and pathologic processes requiring cell migration. Plasminogendependent cell migration is involved in macrophage recruitment during the inflammatory response, 4-10 tissue remodeling, 11 wound healing, 12,13 tumor cell invasion and metastasis, 14,15 skeletal myogenesis, 16 neuroendocrine prohormone processing, 17,18 and neurite outgrowth. 19,20 Studies in plasminogen-deficient mice have demonstrated that plasminogen plays a key role in cell migration in a diverse array of physiologic and pathophysiologic settings, notably, macrophage recruitment in response to inflammatory stimuli in the thioglycollate-induced model of peritonitis. Plasmin-dependent cell migration is accomplished by direct degradation of extracellular matrix components by plasmin and also by activation of matrix metalloproteinases for further degradation of extracellular matrices. [4][5][6][7] Among the plasminogen-binding proteins, those exposing Cterminal basic residues on cell surfaces are predominantly responsible for the ability of eukaryotic cells to enhance plasminogen activation, because carboxypeptidase B (CpB) treatment abrogates cell surface-dependent plasminogen activation. 21 Furthermore, plasminogen-dependent macrophage recruitment is mediated by CpB-sensitive plasminoge...

Corneal infection with Pseudomonas aeruginosa perforates the cornea in susceptible C57BL/6 (B6), but not resistant BALB/c, mice. To determine whether vasoactive intestinal peptide (VIP) played a role in development of the resistant response, protein expression levels were tested by immunocytochemistry and enzyme immunoassay in BALB/c and B6 corneas. Both mouse strains showed constitutive expression of corneal VIP protein and nerve fiber distribution. However, disparate expression patterns were detected in the cornea after infection. VIP protein was elevated significantly in BALB/c over B6 mice at 5 and 7 days postinfection. Therefore, B6 mice were injected with rVIP and subsequently demonstrated decreased corneal opacity and resistance to corneal perforation compared with PBS controls. rVIP- vs PBS-treated B6 mice also demonstrated down-regulation of corneal mRNA and/or protein levels for proinflammatory cytokines/chemokines: IFN-γ, IL-1β, MIP-2, and TNF-α, whereas anti-inflammatory mediators, IL-10 and TGF-β1, were up-regulated. Treatment with rVIP decreased NO levels and polymorphonuclear neutrophil (PMN) number. To further define the role of VIP, peritoneal macrophages (Mφ) and PMN from BALB/c and B6 mice were stimulated with LPS and treated with rVIP. Treatment of LPS-stimulated Mφ from both mouse strains resulted in decreased IL-1β and MIP-2 protein levels; PMN responded similarly. Both cell types also displayed a strain-dependent differential response to rVIP, whereby B6 Mφ/PMN responded only to a higher concentration of VIP compared with cells from BALB/c mice. These data provide evidence that neuroimmune regulation of the cytokine network and host inflammatory cells functions to promote resistance against P. aeruginosa corneal infection.

Studies have shown that after Pseudomonas aeruginosa (P. aeruginosa) corneal infection, BALB/c mice that are capable of resolving the disease, locally produce IFN-c. As T cells are not detected in the infected cornea of these mice, antibody depletion was used to test whether NK cells produce the cytokine. After depletion, decreased corneal IFN-c mRNA and increased disease severity, bacterial load, and PMN infiltrate resulted. Further work determined if substance P (SP), a pro-inflammatory neuropeptide, participated in regulation of this response. To this end, mice were treated with the SP antagonist, spantide I that blocks SP interaction with neurokinin-1, its major receptor. The treatment significantly decreased corneal IFN-c and IL-18 protein levels and corneal perforation resulted. In vitro experiments using isolated splenic NK cells confirmed their ability to respond to IL-18 and SP and to secrete IFN-c protein. We conclude: that for development of the BALB/c resistance response, NK cells are required to produce IFN-c; that the cells express the neurokinin-1 receptor; and that SP directly regulates IFN-c production through this receptor. The data suggest a unique link between the nervous system and development of innate immunity in the cornea.

The SP antagonist Spantide provides a novel approach to reduce type 1 and enhance the type 2 cytokine IL-10 in the infected cornea of B6 mice, leading to a significant reduction in corneal perforation and improved disease outcome.

Summary Background Plg-RKT is a novel integral membrane plasminogen receptor that binds plasminogen via a C-terminal lysine exposed on the cell surface and promotes plasminogen activation on the cell surface by both tissue plasminogen activator and urokinase plasminogen activator. Objectives To evaluate the role of Plg-RKT in vivo we generated Plg-RKT-/- mice using a homologous recombination technique. Methods We characterized the effect of Plg-RKT deletion on reproduction, viability, health, and spontaneous thrombosis and inflammation. Results Plg-RKT-/- mice were viable and fertile. Survival of Plg-RKT-/- mice and Plg-RKT+/+ littermates was not significantly different. However, quite strikingly, all pups of Plg-RKT-/- females died within 2 days of birth, consistent with a lactation defect in Plg-RKT-/- mothers. Additionally, there was a significant effect of Plg-RKT deficiency on growth rates of female, but not male mice. In experimental peritonitis studies, Plg-RKT-/- mice exhibited a marked defect in macrophage recruitment. As a contributing mechanism, the capacity of Plg-RKT-/- macrophages for plasminogen binding was markedly decreased. Conclusions These studies demonstrate that Plg-RKT is required for plasminogen binding and macrophage migration in vivo. In addition, Plg-RKT deficiency is not compatible with survival of the species, due to death of all offspring of Plg-RKT-/- females. This new mouse model will be important for future studies aimed at delineating the role of cell surface plasminogen activation in challenge and disease models in vivo.