doi:10.1016/j.clim.2004.07.005 
Copyright © 2004 Elsevier Inc. All rights reserved.
The Th2 response as monitored by CRTH2 or CCR3 expression is severely decreased during septic shock
Fabienne Venet, Alain Lepape, Anne-Lise Debard, Jacques Bienvenu, Julien Bohé and Guillaume Monneret
, 
Flow Cytometry Unit, Immunology Laboratory and Intensive Care Units, Lyon-Sud University Hospital, CEDEX F-69495 Pierre-Bénite, France
Received 25 May 2004;
accepted 8 July 2004.
Available online 16 September 2004.
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Abstract
The shift of T lymphocytes toward a Th2 profile during septic shock has been established on the basis of in vitro cytokine production. In the present study, the Th2 response was investigated at the level of cell surface marker expression (whole blood flow cytometry). In 58 patients with septic shock, we observed a reduced CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) expression on Th2 lymphocytes and regulatory T cells in comparison with 39 healthy volunteers. Eosinophils, which constitutively express CRTH2 in healthy individuals, also exhibited low levels of CRTH2 in patients. In addition, eosinophil CCR3 expression (eotaxin receptor, type 2 chemokine) was strongly correlated with CRTH2, suggesting thus an extended modulation of Th2 related molecules. Importantly, the persistence over time of low levels of CRTH2 or CCR3 expression was found in nonsurvivors. We hypothesize that the restoration of CRTH2/CCR3 expression may be an indicator for optimal recovery after septic shock.
Keywords: Septic shock; CRTH2; Th2; Regulatory T cells; Eosinophils; Immunoparalysis
Fig. 1. Identification of CRTH2 expressing cells by flow cytometry. (A) Ungated leukocytes biparametric representation on the basis of side scatter characteristics (SSC, y-axis) and FITC-CRTH2 (x-axis). Two CRTH2 expressing cell populations are easily distinguishable: the one with high light scatterings corresponds to the eosinophil population; the second one (gating region) comprises Th2 lymphocytes and basophils. (B) Cells from the gating region expressed on the basis of PE-CD4 (x-axis) and ECD-CD3 (y-axis) characteristics. Th2 lymphocytes were separated from basophils based on their expression of both CD3 and CD4. We also confirmed that the remaining cells (CD3 and CD4 negative) all expressed CD203c, a specific marker of basophils (data not shown). (C) Ungated leukocytes monoparametric representation on the basis of CRTH2 expression. The threshold for positivity (i.e., CRTH2 expressing cells) is easily set up between the two peaks of fluorescence.
Fig. 2. CRTH2 and CCR3 expressions are closely correlated on eosinophils. Eosinophils from 39 septic patients (open squares) and 22 healthy individuals (filled squares) were examined for the expression of CRTH2 and CCR3. Septic patients were monitored between days 1–2 and day 15 after the onset of shock (all observation days are plotted, n = 127). Linear regression analyses demonstrated a significant correlation between these percentages.
Fig. 3. CRTH2-CCR3 coexpression is severely decreased on eosinophils from septic patients. Eosinophils from 39 septic patients and 22 healthy individuals were examined for the coexpression of CRTH2 and CCR3. Septic patients were monitored between days 1–2 and day 15 after the onset of shock. Data are expressed as median, 25th–75th percentile (Q1–Q3) are given below the graph for each time point. For determining normal values, eosinophils from healthy individuals were phenotyped once: median: 93.3, (86.1–96.4). *P < 0.001 (Mann Whitney).
Table 1.
Demographic and clinical characteristics
* Mean (95% CI).
# P < 0.01 vs. survivors.
Table 2.
CRTH2 expression on lymphocytes
Whole blood CD4+ lymphocytes and regulatory T cells (Treg) from septic patients (n = 33, at day 5 after the onset of shock) and from healthy donors (n = 30) were examined for CRTH2 expression. Results are expressed as median and 25th–75th percentile (Q1–Q3). The nonparametric Mann–Whitney test was used to assess significance between groups.
* P < 0.05 against corresponding values in septic patients.
** P < 0.01 against corresponding values in septic patients.
Table 3.
CRTH2 and CCR3 expressions on eosinophils
Eosinophils (VLA-4+ granulocytes) were examined for the mean fluorescence intensity (MFI) of CRTH2 and CCR3 both in septic patients (n = 32, at day 5 after the onset of shock) and in healthy donors (n = 22). The threshold for CRTH2 and CCR3 positivity was easily set up on a monoparametric histogram between cell populations expressing these markers or not (see Fig. 1C as an example). Results are presented as median and 25th–75th percentile (Q1–Q3). The nonparametric Mann–Whitney test was used to assess significance between groups.
Table 4.
CRTH2 and CCR3 expressions on eosinophils from septic patients
Eosinophils from 39 septic patients (27 survivors and 12 nonsurvivors) were examined for the coexpression of CRTH2 and CCR3 during the 15 days of the following. Results are expressed as median and 25th–75th percentile (Q1–Q3).
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