Intravascular monocyte populations following lupus nephritis and acute NTN, analysis of isolated monocytes for adoptive transfer, analysis of renal intravascular monocyte accumulation in Tnfr KO, in vivo CD45 antibody controls, and effect of blocking adhesion on monocyte acquisition of MHCII and F4/80. (A and B) Intravascular monocyte populations were determined in NZB/W lupus-prone mice with accelerated nephritis (A) and Wt mice subjected to acute NTN (B) using approaches described in Fig. 5. (C) Left: Monocytes isolated from the bone marrow of Wt and Ccr2 KO mice were examined for Ly6C and MHCII. All cells were Ly6C⁺, and >95% were Ly6C⁺MHCII⁻ (P1) and <1.5% were Ly6C⁺MHCII⁺ (P2). Middle: The two Ly6C⁺ populations were further analyzed for CX3CR1 and CCR2. The majority of Ly6C⁺MHCII⁻ (P1) cells were CCR2⁺ and CX3CR1⁻. Right: Monocytes from isolated bone marrow of Wt and Ccr2 KO were also examined for Ly6C and F4/80. All cells were Ly6C⁺: >95% were Ly6C⁺F4/80⁻ and ≤0.5% were Ly6C⁺F4/80⁺. (D) Wt and TNFR2-deficient (Tnfr2 KO) mice subjected to NTN received CellTrace CFSE dye (AF488)–labeled Wt and CellTrace violet dye (BV421)–labeled Ccr2 KO monocytes i.v., and the frequencies of the labeled monocytes in blood and kidney were calculated. (E) Intravascular accumulation of leukocytes in lymph nodes and brain of mice subjected to acute NTN. Data are expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA with Dunnett’s t test correction. (F) Monocytes treated with isotype (Iso.) or functional blocking antibody to CD18 (aCD18) or VLA-4 (aVLA-4) were incubated with endothelial cells or endothelial cells treated with TNF (EC + TNF). Adherent monocytes were evaluated for acquisition of MHCII (left) or F4/80 (right). *, P < 0.05; **, P < 0.01; ***, P < 0.005.