Lymphocyte-associated MMP-9 is activated by lymphocyte binding to VCAM-1. Mouse spleen cells were isolated, and red blood cells were removed by hypotonic shock. (A) mHEVc cells were incubated with spleen cells at 37° C. Nonbound cells were removed after 15 minutes because this yielded maximal cell adhesion.⁴  At the times indicated, bound lymphocytes were collected by reversing their binding with blocking anti–VCAM-1 antibody³  and washed. Equal cell equivalents were examined by zymography as described in Figure 2. (B) Spleen cells were incubated with 1 μM H₂O₂ at 37° C. (C) mHEVc cells were pretreated with apocynin (4 mM) for 30 minutes. Nonbound cells were removed after 15 minutes. Bound lymphocytes were recovered and analyzed as described for panel A. (D) mHEVc cells or lymphocytes were pretreated with diphenyliodonium (DPI; 5 μM) for 30 minutes and washed prior to coculture with nontreated (NT) lymphocytes or nontreated mHEVc cells, respectively. Nonbound cells were removed after 15 minutes of coculture. Bound lymphocytes were recovered and analyzed as described for panel A. (E-H) Lymphocytes were incubated with 1 μMH₂O₂ as in panel B. Western blots were performed with equal cell number by using mouse antimouse TIMP-1 (clone 102B1) or TIMP-2 (clone 67-4H11) antibodies (1:1000; Oncogene, EMD Biosciences, La Jolla, CA) or a rabbit antimouse MMP-9 antibody (1:500; Biomol). (E) TIMP-1. (F) TIMP-2. (G) MMP-9. (H) MMP-9/TIMP ratio. Data presented are the mean ± SD from 2 experiments. *P < .05 compared with nonstimulated lymphocytes (0 hours). **P < .05 compared with nontreated lymphocytes at 5 hours.