Erythromycin exerts in vivo anti-inflammatory activity downregulating cell adhesion molecule expression

Abstract

1. Macrolides have long been used as anti-bacterial agents; however, there is some evidence that may exert anti-inflammatory activity. Therefore, erythromycin was used to characterize the mechanisms involved in their in vivo anti-inflammatory activity. 2. Erythromycin pretreatment (30 mg kg(-1) day(-1) for 1 week) reduced the lipopolysaccharide (LPS; intratracheal, 0.4 mg kg(-1))-induced increase in neutrophil count and elastase activity in the bronchoalveolar lavage fluid (BALF) and lung tissue myeloperoxidase activity, but failed to decrease tumor necrosis factor-alpha and macrophage-inflammatory protein-2 augmented levels in BALF. Erythromycin pretreatment also prevented lung P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA upregulation in response to airway challenge with LPS. 3. Mesentery superfusion with LPS (1 mug ml(-1)) induced a significant increase in leukocyte-endothelial cell interactions at 60 min. Erythromycin pretreatment abolished the increases in these parameters. 4. LPS exposure of the mesentery for 4 h caused a significant increase in leukocyte rolling flux, adhesion and emigration, which were inhibited by erythromycin by 100, 93 and 95%, respectively. 5. Immunohistochemical analysis showed that LPS exposure of the mesentery for 4 h caused a significant enhancement in P-selectin, E-selectin, ICAM-1 and VCAM-1 expression that was downregulated by erythromycin pretreatment. 6. Flow cytometry analysis indicated that erythromycin pretreatment inhibited LPS-induced CD11b augmented expression in rat neutrophils. 7. In conclusion, erythromycin inhibits leukocyte recruitment in the lung and this effect appears mediated through downregulation of CAM expression. Therefore, macrolides may be useful in the control of neutrophilic pulmonary diseases.

Figure 1

Effect of erythromycin on LPS-induced leukocyte recruitment in rat lung. Neutrophil counts (a) and elastase activity (b) in the BALF and lung tissue MPO activity (c) in the following experimental groups: untreated rats challenged with saline (negative control); untreated rats exposed to LPS (0.4 mg kg⁻¹, i.t.; ∼100 μg rat⁻¹) (positive control); and saline- or LPS-exposed rats pretreated with erythromycin (30 mg kg⁻¹ day⁻¹ for 1 week before challenge). BALF for counting of neutrophils and determination of elastase activity was obtained at 10 h postexposure to saline or LPS. The lung tissue samples for determination of MPO activity were obtained at 4 h postsaline or LPS exposure. Data are mean±s.e.m. of 10 (negative control, that is, vehicle+saline), 14 (positive control, that is, vehicle+LPS), 5 (erythromycin+saline) and 15 (erythromycin+LPS) animals in each group; ^*P<0.05 compared to negative control; ⁺P<0.05 compared to positive control.

Figure 2

Effect of erythromycin on LPS-induced release of TNF-α (a) and MIP-2 (b) in BALF. TNF-α and MIP-2 levels in BALF of rats in the following experimental groups: untreated rats challenged with saline (negative control); untreated rats exposed to LPS (0.4 mg kg⁻¹, i.t.; ∼100 μg rat⁻¹) (positive control); and saline- or LPS-exposed rats pretreated with erythromycin (30 mg kg⁻¹ day⁻¹ for 1 week before challenge). BALF was obtained at 4 h postexposure to saline or LPS. Data are mean±s.e.m. of 10 (negative control, that is, vehicle+saline), 14 (positive control, that is, vehicle+LPS), 5 (erythromycin+saline) and 15 (erythromycin) rats per group; ^*P<0.05 compared to negative control.

Figure 3

Effect of erythromycin on LPS-induced upregulation of cell adhesion molecule expression in the lung tissue. Relative quantification of the mRNA levels of P-selectin (a), E-selectin (b), ICAM-1 (c), VCAM-1 (d) and GAPDH by real-time quantitative RT–PCR using the comparative C_t method (ΔΔC_t method). Data from five independent experiments are shown for the following experimental groups: vehicle+saline, vehicle+LPS, erythromycin+saline and erythromycin+LPS. Measurements are made at 4 h after challenge with LPS or saline. The C_t values for GAPDH were similar in the different samples, thus confirming the value of this housekeeping gene as endogenous control. Columns show the fold-increase in the expression of the cell adhesion molecules relative to control GAPDH values as mean±s.e.m. of five experiments in each group; ^*P<0.05 compared to vehicle+saline; ⁺P<0.05 from vehicle+LPS.

Figure 4

Effect of erythromycin on acute LPS-induced leukocyte rolling flux (a), leukocyte rolling velocity (b), leukocyte adhesion (c) and leukocyte emigration (d) in rat mesenteric postcapillary venules. Parameters were measured 0, 15, 30 and 60 min after superfusion with buffer or with LPS (1 μg ml⁻¹) in the following experimental groups: untreated rats exposed to buffer (negative control); untreated rats exposed to LPS (positive control); and saline- or LPS-exposed rats pretreated with erythromycin (30 mg kg⁻¹ day⁻¹ for 1 week before superfusion). Data are mean±s.e.m. of 5 (negative control, that is, vehicle+buffer), 9 (positive control, that is, vehicle+LPS), 5 (erythromycin+buffer) and 8 (erythromycin+LPS) rats per group; ^*P<0.05 or ^**P<0.01 compared to negative control; ⁺P<0.05 or ⁺⁺P<0.01 compared to positive control.

Figure 5

Effect of erythromycin on subacute LPS-induced leukocyte rolling flux (a), leukocyte rolling velocity (b), leukocyte adhesion (c) and leukocyte emigration (d) in rat mesenteric postcapillary venules. Parameters were measured 4 h after i.p. injection of 5 ml of saline or 5 ml of LPS (0.2 μg ml⁻¹) in the following experimental groups: untreated rats exposed to buffer (negative control); untreated rats exposed to LPS (positive control); and saline- or LPS-exposed rats pretreated with erythromycin (30 mg kg⁻¹ day⁻¹ for 1 week before LPS injection). Data are mean±s.e.m. of 7 (negative control, that is, vehicle+saline), 7 (positive control, that is, vehicle+LPS), 5 (erythromycin+saline) and 9 (erythromycin+LPS) rats per group; ^**P<0.01 compared to negative control; ⁺⁺P<0.01 compared to positive control.

Figure 6

Representative photomicrographs of rat mesenteric venules showing immunolocalization of P-selectin and ICAM-1 expression in animals untreated and pretreated with erythromycin after acute LPS superfusion. P-selectin expression after buffer (a) and LPS 60 min superfusion in the untreated group (b) and erythromycin-pretreated group (c). ICAM-1 expression after buffer (d) and LPS 60 min superfusion in the untreated group (e) and erythromycin-pretreated group (f). Brown reaction product indicates positive immunoperoxidase localization for both CAMs on the vascular endothelium. All six panels are lightly counterstained with hematoxylin and have the same magnification ( × 400). Results are representative of n=5–6 experiments with each treatment.

Figure 7

Representative photomicrographs of rat mesenteric venules showing immunolocalization of P-selectin, E-selectin, ICAM-1 and VCAM-1 expression in animals untreated and pretreated with erythromycin after subacute LPS exposure. P-selectin expression after 4 h saline (a) or LPS exposure in the untreated group (b) and erythromycin-pretreated group (c). E-selectin expression after 4 h saline (d) or LPS exposure in the untreated group (e) and erythromycin-pretreated group (f). ICAM-1 expression after 4 h saline (g) or LPS exposure in the untreated group (h) and erythromycin-pretreated group (i). VCAM-1 expression after 4 h saline (j) or LPS exposure in the untreated group (k) and erythromycin-pretreated group (l). Brown reaction product indicates positive immunoperoxidase localization for all CAMs on the vascular endothelium. All panels are lightly counterstained with hematoxylin and have the same magnification ( × 400). Results are representative of n=6–7 experiments with each treatment.

Figure 8

Effect of erythromycin on surface expression of α_Mβ₂-integrins (CD11b/CD18) on peripheral rat neutrophils. Rats were untreated or pretreated with erythromycin (30 mg kg⁻¹ day⁻¹ for 1 week) and 1 h later blood samples were collected. Samples were incubated for 4 h with either vehicle (saline) or 1 ng ml⁻¹ LPS, and then stained for 20 min with conjugated mAb and analyzed by flow cytometry. FITC fluorescence values are expressed as the percentage of mean fluorescence intensity of basal values (dotted line). Data are mean±s.e.m. of n=4 experiments. ^*P<0.05 compared to the mean fluorescence intensity in the vehicle-treated group.