Figure 1.
Concentration-dependent effects of glucocorticoids on gene expression in equine peripheral blood neutrophils.
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).
Figure 2.
Glucocorticoid effects on gene expression in equine peripheral blood neutrophils and neutrophil-depleted leukocytes.
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).
Figure 3.
Glucocorticoid effects on pro-inflammatory cytokines gene expression in human peripheral blood neutrophils and neutrophil-depleted leukocytes.
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).
Figure 4.
Comparison of glucocorticoid inhibitory effects between equine cell populations.
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).
Figure 5.
Comparison of dexamethasone inhibitory effect in equine and human neutrophils.
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).
Figure 6.
Glucocorticoids increase survival in equine peripheral blood neutrophils.
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).