Endothelial Specific Molecule 1 (Esm‐1) Inhibits Macrophage Infiltration in a Mouse Model of Goodpasture's Syndrome

Abstract

Goodpasture's syndrome is an autoimmune disorder caused by pathogenic antibodies to kidney and lung basement membranes causing acute kidney and lung injury, respectively. We modeled this syndrome by treatment with anti‐GBM (Glomerular Basement Membrane) antibodies leading to glomerular and tubulointerstitial inflammation in mice. Endothelial Specific Molecule1 (Esm‐1)/endocan is a secreted glycoprotein highly expressed in kidney glomeruli and lung endothelium and is proposed to disrupt molecular interactions that mediate leukocyte infiltration and anti‐GBM nephritis. To discover the in vivo functional role of Esm‐1 in leukocyte infiltration in anti‐GBM mice, we delivered Esm‐1 plasmid for stable integration to anti‐GBM mice and controls by hydrodynamic tail vein injection. We quantified kidney leukocyte infiltration by separating glomerular and tubulointerstitial compartments, dissociating into single cells, and measuring leukocyte subtypes by flow cytometry. In contrast to i.p. administration of recombinant Esm‐1 which has an abbreviated half‐life in mouse serum (less than 2 hours), we induced ~12‐fold increase in circulating Esm‐1 that persists for 2 weeks compared to control injected mice, by hydrodynamic injection of an Esm‐1‐expressing plasmid (11242 +/− 3324 vs. 958 +/− 188 pg/mL, p<0.05, N=3–5/group). We demonstrate that by flow cytometry, anti‐GBM mice show significantly increased glomerular (3.4 +/− 0.47% vs. 1.1 +/− 0.33%, p < 0.01, N=8/group) and tubulointerstitial (1.4 +/− 0.14% vs. 0.1+/− 0.032%, p < 0.01, N=8/group) CD45+ leukocytes seven days post treatment with anti‐GBM antisera. The increase in CD45+ cells includes increased glomerular (3.4 +/− 0.47% vs. 1.1 +/− 0.33%, p < 0.01) and tubular (1.2 +/− 0.14% vs. 0.1+/− 0.025%, p < 0.01) macrophages. By increasing circulating Esm‐1, we significantly decreased macrophage infiltration in the tubulointerstitium (4.2 ± 1.3% vs. 1.6 ± 0.4%, P < 0.01), but did not influence glomerular macrophage infiltration (2.3 ± 1.2% vs. 1.5 ± 0.2%, P = 0.13). Furthermore, Esm‐1 does not alter kidney neutrophils (glomerular: 0.013 ± 0.007% vs. 0.010 ± 0.006%, p = 0.61; tubulointerstitial: 0.19 ± 0.02% vs. 0.27 ± 0.07%, p = 0.085) or lymphocytes(glomerular: 1.7 ± 0.8% vs. 1.9 ± 0.9%, p = 0.89; tubulointerstitial: 1.9 ± 0.70% vs. 2.2 ± 0.71%, p = 0.64). We also found that inhibition of macrophage infiltration was dependent on glycosylation of Esm‐1. Furthermore, we have generated Esm‐1 knockout mice for complementary studies. We conclude that in contrast to deletion of the inflammatory mediator ICAM‐1, Esm‐1 inhibits inflammation in a tissue and cell‐specific manner. Esm‐1 provides a natural biologic inhibitor of inflammation, and based on its expression pattern, may be a key therapeutic target in pulmonary‐renal syndromes.Support or Funding Information1. Larry L. Hillblom Foundation2. Holmgren Family3. NIDDK