Neutrophils Are Not Less Sensitive Than Other Blood Leukocytes to the Genomic Effects of Glucocorticoids

<div><h3>Background</h3><p>Neutrophils are generally considered less responsive to glucocorticoids compared to other inflammatory cells. The reported increase in human neutrophil survival mediated by these drugs partly supports this assertion. However, it was recently shown that dexamethasone exerts potent anti-inflammatory effects in equine peripheral blood neutrophils. Few comparative studies of glucocorticoid effects in neutrophils and other leukocytes have been reported and a relative insensitivity of neutrophils to these drugs could not be ruled out.</p> <h3>Objective</h3><p>We assessed glucocorticoid-responsiveness in equine and human peripheral blood neutrophils and neutrophil-depleted leukocytes.</p> <h3>Methods</h3><p>Blood neutrophils and neutrophil-depleted leukocytes were isolated from 6 healthy horses and 4 human healthy subjects. Cells were incubated for 5 h with or without LPS (100 ng/mL) alone or combined with hydrocortisone, prednisolone or dexamethasone (10⁻⁸ M and 10⁻⁶ M). IL-1β, TNF-α, IL-8, glutamine synthetase and GR-α mRNA expression was quantified by qPCR. Equine neutrophils were also incubated for 20 h with or without the three glucocorticoids and cell survival was assessed by flow cytometry and light microscopy on cytospin preparations.</p> <h3>Results</h3><p>We found that glucocorticoids down-regulated LPS-induced pro-inflammatory mRNA expression in both cell populations and species. These drugs also significantly increased glutamine synthetase gene expression in both equine cell populations. The magnitude of glucocorticoid response between cell populations was generally similar in both species. We also showed that dexamethasone had a comparable inhibitory effect on pro-inflammatory gene expression in both human and equine neutrophils. As reported in other species, glucocorticoids significantly increase the survival in equine neutrophils.</p> <h3>Conclusions</h3><p>Glucocorticoids exert genomic effects of similar magnitude on neutrophils and on other blood leukocytes. We speculate that the poor response to glucocorticoids observed in some chronic neutrophilic diseases such as severe asthma or COPD is not explained by a relative lack of inhibition of these drugs on pro-inflammatory cytokines expression in neutrophils.</p> </div

Glucocorticoids increase survival in equine peripheral blood neutrophils.

<p>A. Freshly isolated equine neutrophils (n = 6) were incubated for 20 h in the absence or presence of hydrocortisone (HC), prednisolone (PRED), dexamethasone (DEX) at 10⁻⁶ M and 10⁻⁸ M. The number of viable cells was assessed by flow cytometry using APC Annexin V/7-AAD staining. APC Annexin V and 7-AAD negative cells were considered as viable cells. Bars represent means + SEM. *Significant effect of treatment over unstimulated cells (p≤0.001). B. The percentage of viable cells was assessed by flow cytometry using APC Annexin V/7-AAD staining (x axis) and by light microscopy (y axis) and analyzed using a simple linear regression (linear regression slope R² = 0.67, p<0.0001).</p

Comparison of glucocorticoid inhibitory effects between equine cell populations.

<p>Neutrophils and neutrophil depleted leukocytes were isolated from the blood of 6 healthy horses and were incubated for 5 h with or without lipopolysaccharide (LPS; 100 ng/mL) alone or combined with hydrocortisone (HC), prednisolone (PRED), dexamethasone (DEX) at 10⁻⁶ M. Following culture, mRNA expression of pro-inflammatory cytokines (IL-1β and IL-8; A and B) was quantified by qPCR. Absolute values were corrected using GAPDH as a reference gene. GC percentage of inhibition on LPS-induced mRNA expression was compared between cell populations. A 100% inhibition means that mRNA expression returned to unstimulated cell basal mRNA level. Bars represent means + SEM. *Significant effect of treatment between cell populations (p = 0.046).</p

Concentration-dependent effects of glucocorticoids on gene expression in equine peripheral blood neutrophils.

<p>Neutrophils were isolated from the blood of 3 healthy horses and were incubated for 5 h with or without lipopolysaccharide (100 ng/mL) combined with hydrocortisone (HC), prednisolone (PRED), dexamethasone (DEX) at 10⁻⁸ M and 10⁻⁶ M. Following culture, mRNA expression of pro-inflammatory cytokines (IL-1β, TNF-α and IL-8; A, B and C), glutamine synthetase (D) and GR-α (E) was quantified by qPCR. Absolute values were corrected using GAPDH as a reference gene. Gene expression was reported as the relative variation (fold increase) to unstimulated cell mRNA levels (arbitrary value of 1). Bars represent means. *Significant difference between treatments (p≤0.020).</p

Glucocorticoid effects on gene expression in equine peripheral blood neutrophils and neutrophil-depleted leukocytes.

<p>Neutrophils and neutrophil-depleted leukocytes were isolated from the blood of 6 healthy horses and were incubated for 5 h with or without lipopolysaccharide (LPS; 100 ng/mL) alone or combined with hydrocortisone (HC), prednisolone (PRED), dexamethasone (DEX) at 10⁻⁶ M. Following culture, mRNA expression of pro-inflammatory cytokines (IL-1β, TNF-α and IL-8; A, B and C), glutamine synthetase (D) and GR-α (E) was quantified by qPCR. Absolute values were corrected using GAPDH as a reference gene. Gene expression was reported as the relative variation (fold increase) to unstimulated cell mRNA levels (arbitrary value of 1). Bars represent means + SEM. *Significant effect of treatment over LPS-stimulated cells (p≤0.014). †Significant difference between treatments (p = 0.009).</p

Glucocorticoid effects on pro-inflammatory cytokines gene expression in human peripheral blood neutrophils and neutrophil-depleted leukocytes.

<p>Neutrophils and neutrophil depleted leukocytes were isolated from the blood of 4 healthy human volunteers and were incubated for 5 h with or without lipopolysaccharide (LPS; 100 ng/mL) alone or combined with dexamethasone (DEX) at 10⁻⁶ M. Following culture, mRNA expression of proinflammatory cytokines (IL-1β, TNF-α and IL-8) was quantified by qPCR. Absolute values were corrected using GAPDH as a reference gene. A. Gene expression was reported as the relative variation (fold increase) to unstimulated cell mRNA levels (arbitrary value of 1). Bars represent means. *Significant effect of treatment over LPS-stimulated cells (p≤0.050). B. Dexamethasone percentage of inhibition on LPS-induced mRNA expression was compared between cell populations. A 100% inhibition means that mRNA expression returned to unstimulated cell basal mRNA level. Bars represent means. †Significant effect of treatment between cell populations (p = 0.048).</p

Comparison of dexamethasone inhibitory effect in equine and human neutrophils.

<p>Neutrophils were isolated from the blood of 6 healthy horses and 4 healthy human volunteers and were incubated for 5 h with or without lipopolysaccharide (LPS; 100 ng/mL) alone or combined with dexamethasone (DEX) at 10⁻⁶ M. Following culture, mRNA expression of pro-inflammatory cytokines (IL-1β, TNF-α and IL-8; A, B and C) was quantified by qPCR. Absolute values were corrected using GAPDH as a reference gene. Dexamethasone percentage of inhibition on LPS-induced mRNA expression was compared between both species. A 100% inhibition means that mRNA expression returned to unstimulated cell basal mRNA level. Bars represent means + SEM. *Significant effect of treatment between cell populations (p = 0.013).</p

Methodology for SSH.

<p>Schematic representation of the different steps described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029440#s2" target="_blank">Material and Methods</a> (A), the sample pooling (n corresponds to the number of samples) (B), and the different hybridization steps performed with the SSH technique (C).</p

Differential hybridization screening.

<p>Representative differential screening results of macroarrays of the SH-Ctls library. Four identical membranes were dot-blotted with PCR products obtained by SSH. The membranes were then hybridized with four different probes: SH-Ctls subtracted cDNAs (A), SH unsubtracted cDNAs (B), Ctls-SH subtracted cDNAs (C) and Ctls unsubtracted cDNAs. The arrow in the top left corner indicates the positive control (<i>LCN2</i>). The arrow head indicates an example of differentially expressed genes in SH compare with Ctls.</p

Evaluation of subtraction efficiency.

<p>A: Reduction of <i>GAPDH</i> cDNA following subtraction in the SH-Ctls sample. PCR was performed on SH-Ctls subtracted and SH unsubtracted samples. <i>GAPDH</i> PCR products (760 pb) were detectable 10 cycles earlier in the unsubtracted sample (18 cycles) than in the subtracted sample (28 cycles). B: Enrichment of <i>LCN2</i> cDNA following subtraction in the SH-Ctls sample. PCR was performed on SH-Ctls and Ctls-SH subtracted samples as well as SH and Ctls unsubtracted samples. <i>LCN2</i> PCR products (210 pb) were detected after 20 cycles for both SH unsubtracted and SH-Ctls subtracted samples, the difference in the intensity of the 2 bands indicate the enrichment compare to Ctls unsubtracted and Ctls-SH subtracted samples.</p