Differential roles of selectins and the alpha4-integrin in acute, subacute, and chronic leukocyte recruitment in vivo.

Adhesion blocking mAbs specific for rat P-, E-, and L-selectin and the alpha4-integrin were used to characterize leukocyte recruitment mechanisms in models of LTC4 (acute), LPS (subacute), and adjuvant-induced (chronic) inflammation. Intravital microscopy was employed to measure leukocyte rolling and adhesion in rat mesenteric venules. Superfusing the mesentery with 20 nM LTC4 elicited an increase in leukocyte rolling (66.8 +/- 3.8 vs 18.2 +/- 3.2 cells/min control) that was completely eliminated by an anti-rat P-selectin mAb. Superfusion with 1 microg/ml LPS induced a significant increase in leukocyte rolling within 15 min (73 +/- 8 vs 33 +/- 6 cells/min control). Rolling increased further starting at 105 min and peaked by 150 min (141 +/- 23 cells/min). LPS-induced leukocyte rolling was eliminated during the first 90 min by the P-selectin mAb. The later increase in leukocyte rolling was not prevented by a second treatment with P-selectin mAb or a function-blocking mAb against rat E-selectin. This later phase of leukocyte rolling was blocked by treatments with mAbs against either the alpha4-integrin or L-selectin. Twelve days following Mycobacterium butyricum immunization, 300 to 500 rolling cells/min were observed. This could be reduced approximately 50 to 60% by mAb against either the alpha4-integrin or L-selectin. The combination of both mAbs eliminated approximately 90% of rolling. Neither the P- nor E-selectin mAbs reduced rolling in this chronic inflammatory model. This study highlights differences in leukocyte adhesive mechanisms elicited by different stimuli and at different time points within the same vascular bed.

Generation and characterization of a novel adhesion function blocking monoclonal antibody recognizing both rat and mouse E-selectin.

The prerequisite for the recruitment of circulating leukocytes to sites of inflammation is adhesion to vascular endothelial cells. Selectins play a significant role in the initiation of this multistep process by mediating "rolling" of the leukocytes on the endothelium. Investigation of selectin-dependent cell interactions using function blocking monoclonal antibodies (MAb) provides insights into the mechanisms involved in leukocyte migration into inflammation. Until now most studies in inflammation models in rats have relied on cross-reactive or polyclonal antibodies against rat E-selectin. In an E-selectin knockout mouse, we aimed to generate an adhesion function blocking MAb to rat E-selectin by immunization with rat E-selectin transfected Chinese hamster ovary cells (RESEC). An IgG1 kappa MAb was identified that reacts with RESEC but not with untransfected Chinese hamster ovary cells, as well as with recombinant mouse E-selectin protein as assessed by ELISA. This MAb is designated RME-1. It does not cross-react with rat L-selectin or rat P-selectin or E-selectin expressed on human umbilical vein endothelium. Adhesion of the HL-60 myeloid cells to immobilized mouse E-selectin was completely inhibited by MAb RME-1 under static conditions and adhesion of rat polymorphonuclear leukocytes to recombinant mouse E-selectin was blocked under rotation condition. This novel antibody thus recognizes a function-related epitope on rodent E-selectin.

The role of E- and P-selectin in neutrophil and monocyte migration in adjuvant-induced arthritis in the rat.

The role of the endothelial adhesion molecules E- and P-selectin in leukocyte accumulation in arthritis is not known. We investigated this role in rat adjuvant arthritis by employing adhesion function-blocking monoclonal antibodies (mAb) to rat P- and E-selectin. The acute migration (2 h) of radiolabeled rat blood neutrophils and monocytes to joints and skin was determined. Anti-P-selectin mAb significantly reduced accumulation of monocytes (by 50%) and neutrophils (by 40%) in the talar joint, and of neutrophils in tail joints (by 90%). Anti-E-selectin mAb alone did not attenuate leukocyte migration, but when combined with anti-P-selectin mAb, it enhanced inhibition of neutrophil accumulation in the talar and carpal joints. In the same animals, anti-P-selectin mAb significantly inhibited neutrophil and monocyte migration to dermal inflammatory reactions induced by zymosan-activated rat serum (ZAS) containing the chemotactic factor C5ades Arg, endotoxin (LPS), interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). In contrast, anti-E-selectin mAb alone had no effect on monocyte or neutrophil accumulation in inflamed skin of arthritic animals, but again enhanced the inhibition when combined with mAb to P-selectin. The addition of anti-L-selectin mAb to anti-P- and E-selectin mAb did not further suppress monocyte or neutrophil migration to inflamed skin or joints. These results demonstrate that optimal leukocyte migration to arthritic joints and inflamed skin is P-selectin dependent, and E-selectin is not essential. However, E-selectin contributes to migration when P-selectin mechanisms are not operative. L-selectin does not play a role in E- and P-selectin-independent leukocyte migration to joints or skin inflammation in arthritic rats. However, it is likely that additional selectin-independent pathways also mediate neutrophil and monocyte migration to joint and skin inflammation.

Characterization of a novel adhesion function blocking monoclonal antibody to rat/mouse P-selectin generated in the P-selectin-deficient mouse.

P-selectin is an important adhesion molecule involved in leukocyte migration. However, to date, no monoclonal antibodies (MAb) generated against rat P-selectin have been identified which block P-selectin mediated leukocyte adhesion. Most studies in the rat have utilized crossreacting antibodies generated against P-selectin in higher species. In a P-selectin deficient mouse we generated an anti-rat/mouse P-selectin MAb, designated RMP-1, by immunization with activated rat platelets. This IgG2a MAb immunoprecipitates a 140 kDa protein under reducing conditions from rat platelet lysate. By ELISA and immunofluorescence flow cytometry, MAb RMP-1 reacts with thrombin-activated but not unactivated rat platelets. In addition, by ELISA MAb RMP-1 binds to activated mouse platelets and recombinant rat and mouse P-selectin. MAb RMP-1 inhibited adhesion of HL-60 myeloid cells to immobilized mouse P-selectin by 97% and to activated rat and mouse platelets by 100% under static conditions, confirming the adhesion function blocking activity of MAb RMP-1. This novel MAb should be useful for studying P-selectin function in vitro and in vivo in both rat and mouse inflammation models.

Role of E- and P-selectin in migration of monocytes and polymorphonuclear leucocytes to cytokine and chemoattractant-induced cutaneous inflammation in the rat.

The contribution of E- and P-selectin in the rat to the migration of polymorphonuclear leucocytes (PMNL) and monocytes to acute dermal inflammation induced by a chemoattractant (C5ades Arg) or endothelial cell activating agents [lipopolysaccharide, tumour necrosis factor-alpha (TNF-alpha), alpha-thrombin and interferon-gamma] administered intradermally was investigated. Migration was quantitated using radiolabelled blood PMNL and monocytes and new, function-blocking monoclonal antibodies (mAbs) to rat E- and P-selectin were employed. Monocyte migration to inflamed skin was partially inhibited (40-75%) by P-selectin mAb with all stimuli tested, but not by anti-E-selectin. PMNL migration in response to all stimuli was also inhibited (50-75%) by blocking P-selectin, but in contrast to monocytes, PMNL accumulation was partially inhibited by mAb to E-selectin in alpha-thrombin and TNF-alpha lesions. When P-selectin was blocked by mAb, mAb to E-selectin significantly inhibited further (by 70-90%) both PMNL and monocyte accumulation in all lesions, indicating that both P- and E-selectin contribute to the migration of these leucocytes. Blocking L-selectin in addition to P- and E-selectin, had no effect on the remaining migration. Thus, optimal PMNL and monocyte migration to chemotactic factor- and cytokine-induced skin inflammation is P-selectin dependent. E-selectin becomes important, in most conditions used here, when P-selectin mediated recruitment is not operative. A selectin independent pathway likely mediates up to 20% of PMNL and monocyte migration to acute inflammation, at least in skin.