Isthmin 1 is a secreted protein expressed in skin, mucosal tissues, and NK, NKT, and th17 cells

Abstract

Using a comprehensive microarray database of human gene expression, we identified that in mammals, a secreted protein known as isthmin 1 (ISM1) is expressed in skin, mucosal tissues, and selected lymphocyte populations. ISM1 was originally identified in Xenopus brain during development, and it encodes a predicted ∼50-kDa protein containing a signal peptide, a thrombospondin domain, and an adhesion-associated domain. We confirmed the pattern of expression of ISM1 in both human and mouse tissues. ISM1 is expressed by DX5(+) lung lymphocytes that include NK and NKT-like cells, and is also expressed by some CD4(+) T cells upon activation but its expression increases significantly when CD4(+) T cells were polarized to the Th17 lineage in vitro. The presence of IFN-γ during CD4(+) T cell polarization inhibits ISM1 expression. Given that ISM1 has been reported to have anti-angiogenic properties, these observations suggest that ISM1 is a mediator of lymphocyte effector functions and may participate in both innate and acquired immune responses.

FIG. 1.

ISM1 is selectively expressed in the human body and upregulated in activated CD4⁺ T cells. (A) Mean expression values (y-axis) from microarray data for 105 human tissues are displayed across the x-axis. CNS, central nervous system; PNS, peripheral nervous system; DS, digestive system and oral mucosa; St., muscle, adipose, skin; Va., heart and blood vessels; RS, respiratory system; E, endocrine organs; Ur., kidney, urethra; RT, reproductive tract (male and female); Imm., immune tissues; Leu., peripheral blood cells (monocytes, B and T cells), Em, embryonic (fetal kidney, brain, and placenta). (B) Tissues with the highest ISM1 expression are shown, based on the average signal intensity values of the corresponding probeset (235182_at) from the BIGE database. The MI signal is the average microarray signal from the replicates for each tissue included in the BIGE database. Values reflect (A). (C) Selected tissues were tested to confirm ISM1 expression by qPCR, both in hISM1 and mISM1. (D) mISM1 was detected by western blot in the supernatants of HEK293 cells transfected with the construct pcDNA3.1⁺/mISM1. (E) ISM1 expression was measured by qPCR in resting or activated mouse naive CD4⁺ lymph node T cells, human PBMCs, or Jurkat cells. A representative experiment (out of 3) is shown in (C) and (D). BIGE, body index of gene expression; hISM1, human isthmin 1; mISM1, mouse isthmin 1; MI, mean intensity; PBMCs; qPCR, quantitative real-time polymerase chain reaction.

FIG. 2.

ISM1 is produced by DX5⁺CD4⁻CD8⁻CD3^low and DX5⁺CD4⁻CD8⁻CD3⁻ lung cells. (A) Fresh lung cells from C57BL/6 mice were obtained following collagenase digestion and stained for CD3, CD8, and CD4, followed by intracellular staining for ISM1. (B) Lung cells were stained for CD3, γδ TCR, and ISM1. (C) Lung cells were assayed for surface staining with anti-CD3, DX5, and anti-NKP46 antibodies followed by intracellular ISM1 staining. Corresponding isotype controls for anti-CD3 (PerCP hamster IgG) and DX-5 (FITC rat IgM) antibodies were used to define the CD3- or DX5-positive cells. (D) Fresh lung cells from RAG^−/− γc^−/− mice were stained as in (C). The percentage of cells corresponding to each FACS quadrant is shown. All FACS analyses were performed on the gated lymphocyte population defined by forward versus side scatter (A). Representative experiments are shown (out of 3).

FIG. 3.

ISM1 expression is associated with Th17 cells and negatively regulated by IFN-γ. (A) Mouse lymph node naive CD4⁺ T cells were cultured under CD4⁺ Th polarizing conditions for 5 days. ISM1 expression was measured under non-restimulated (non-restim) or restimulated (restim) conditions by qPCR. Significance was calculated using the mean and standard deviation of 6 independent experiments. (B) Mouse lymph node naive CD4⁺ T cells were cultured with TGFβ+IL-2 or TGFβ+IL-2+anti-IFN-γ for 4 days. ISM1 expression was measured by qPCR from RNA of non-restimulated or restimulated cells. (C) Analysis of RORγt expression was performed by qPCR as described in (B). Statistics were calculated using Student's t-test from 3 independent experiments. Th, T helper.