Dipeptidyl peptidase IV is a human and murine neutrophil chemorepellent

Abstract

In Dictyostelium discoideum, AprA is a secreted protein that inhibits proliferation and causes chemorepulsion of Dictyostelium cells, yet AprA has little sequence similarity to any human proteins. We found that a predicted structure of AprA has similarity to human dipeptidyl peptidase IV (DPPIV). DPPIV is a serine protease present in extracellular fluids that cleaves peptides with a proline or alanine in the second position. In Insall chambers, DPPIV gradients below, similar to, and above the human serum DPPIV concentration cause movement of human neutrophils away from the higher concentration of DPPIV. A 1% DPPIV concentration difference between the front and back of the cell is sufficient to cause chemorepulsion. Neutrophil speed and viability are unaffected by DPPIV. DPPIV inhibitors block DPPIV-mediated chemorepulsion. In a murine model of acute respiratory distress syndrome, aspirated bleomycin induces a significant increase in the number of neutrophils in the lungs after 3 d. Oropharyngeal aspiration of DPPIV inhibits the bleomycin-induced accumulation of mouse neutrophils. These results indicate that DPPIV functions as a chemorepellent of human and mouse neutrophils, and they suggest new mechanisms to inhibit neutrophil accumulation in acute respiratory distress syndrome.

Figure 1

Superimposition of the predicted structure of AprA with the structure of the α/β hydrolase domain of human DPPIV. The catalytic domains of DPPIV (the α/β hydrolase domain of Protein Data Bank ID 1J2E) and AprA (predicted structure) were superimposed and the catalytic triads highlighted. The α/β hydrolase domain of DPPIV is shown in green and its catalytic triad (Asp⁷⁰⁸, His⁷⁴⁰, and Ser⁶³⁰) is orange. The predicted structure of AprA is shown in cyan and its potential catalytic triad (Asp²⁸⁸, His³¹⁹, and Ser¹⁹⁵) is blue. The β-propeller domain of DPPIV was removed for simplicity because the predicted structure of AprA had no overlap with this domain.

Figure 2

Human neutrophils show biased movement away from rDPPIV. Neutrophil migration was measured in the (A) absence or (B) presence of a 0–1.2 nM rDPPIV gradient. Neutrophils were filmed and tracked (see Methods and Materials) during 10-min periods. Orientation is such that the source of rDPPIV is on the left. Graphs are data from one of three independent experiments (see Supplemental Fig. 1 for the other two experiments). Red dots represent the average center of mass for the ending positions of all cells.

Figure 3

Inhibitors of DPPIV reduce the chemorepulsion of neutrophils away from DPPIV. The forward migration index is shown for a gradient of DPPIV, fMLP, two DPPIV inhibitors, or DPPIV with the inhibitors. Values are means ± SEM (n = 3). *p < 0.05 compared with a gradient of DPPIV (by t test).

Figure 4

Purified neutrophils are chemorepulsed by DPPIV. (A) BSA-RPMI 1640 (2%) or (B) 2% human albumin-RPMI 1640 was incubated with (+) or without (−) 300 ng/ml rDPPIV for 3 h. Time at which samples were taken is given in minutes. Gels were silver stained to identify cleavage products. Molecular masses in kilodaltons are indicated at left, * indicates the position of DPPIV, and the arrow indicates the position of albumin. (C) Isolated neutrophils were further purified by CD244 depletion. Purity was determined by morphology. Values are means ± SEM (n = 7). **p < 0.01, ***p < 0.001 for percentages after depletion versus percentages before depletion (by t test). (D) The forward migration index is shown for a DPPIV gradient on purified neutrophils in 2% human albumin-RPMI 1640 buffer. Values are means ± SEM (n = 7). **p < 0.01 versus values from the control (two-tailed unpaired t test). Eos, eosinophils; Lympho, lymphocytes; Mono, monocytes; PMN, polymorphonuclear neutrophil.

Figure 5

Effect of neutrophil-conditioned media on chemorepulsion. (A) The forward migration index is shown for gradients of conditioned media from DPPIV-treated neutrophils (DPPIV CM), gradients of this material treated with DPPIV inhibitor, and gradients of conditioned media from buffer-treated neutrophils (Buffer CM). Values are means ± SEM (n = 4). *p < 0.05, **p < 0.01 versus values from the control (two-tailed unpaired t test). (B) A Western blot of conditioned media using anti-DPPIV Abs. The position of molecular mass standards (in kilodaltons) is indicated on the left.

Figure 6

DPPIV reduces the number of neutrophils in bleomycin-treated lungs. Mice were treated with 3 U/kg bleomycin by oropharyngeal aspirat ion on day 0. On day 1, mice were treated with oropharyngeal aspiration of 0.9 μg human rDPPIV or an equal volume of buffer. Mice were sacrificed on day 3 and cells were collected by BAL. (A) The total number of cells collected from the BAL. Values are means ± SEM (n = 4 for bleomycin and bleomycin plus DPPIV; n = 3 for saline and saline plus DPPIV). *p < 0.05 (t test) (B) The total number of neutrophils, macrophages, and lymphocytes obtained in the BAL for the above experimental groups. Values are means ± SEM (n = 4 for bleomycin and bleomycin plus DPPIV; n = 3 for saline and saline plus DPPIV). *p < 0.05, **p < 0.01 (by paired one-tailed t test). (C) Lung sections were stained with anti-mouse Ly6G to detect neutrophils. Arrows indicate Ly6G⁺ cells. Scale bars, 50 μm. (D) Counts of Ly6G⁺ cells in lung sections. Values are means ± SEM (n = 4 for bleomycin and bleomycin plus DPPIV; n = 3 for saline and saline plus DPPIV). *p < 0.05 (by paired two-tailed t test. (E) Counts of CD11b, CD11c, CD206, or CD107b (Mac3)-positive cells in lung sections. Values are means ± SEM (n =3). *p < 0.05, **p < 0.01 (by paired two-tailed t test).

Figure 7

DPPIV does not regulate neutrophil survival. Human neutrophils were incubated for 20 h in the presence or absence of TNF-α, IL-8, or DPPIV. Values are means ± SEM. (A) Neutrophils were identified by their forward and side scatter characteristics and the number of propidium iodide⁻ cells were counted by flow cytometry (n = 4). (B) The percentage of annexin V⁺ neutrophils was determined by flow cytometry (n = 3). (C) The percentage of apoptotic neutrophils was assessed by counting nuclei with apoptotic morphology on cytospin preparations (n = 3). *p < 0.05 (one-way ANOVA). (D) Counts of activated caspase-3 in lung sections (n = 3).