Effect of tumour necrosis factor-alpha receptor 1 genetic deletion on carrageenan-induced acute inflammation: a comparison with etanercept

Abstract

In the present study, we used tumour necrosis factor-alpha receptor 1 knock-out mice (TNF-alphaR1KO) to evaluate an in vivo role of TNF-alphaR1 on the pathogenesis of inflammatory diseases. We used a murine model of carrageenan-induced acute inflammation (pleurisy), a preclinical model of airway inflammation. The data proved that TNF-alphaR1KO were resistant to carrageenan-induced acute inflammation compared with TNF-alpha wild-type mice. TNF-alphaR1KO showed a significant reduction in accumulation of pleural exudate and in the number of inflammatory cells, in lung infiltration of polymorphonuclear leucocytes and lipid peroxidation and showed a decreased production of nitrite/nitrate in pleural exudates. Furthermore, the intensity and degree of the adhesion molecule intercellular adhesion molecule-1 and P-selectin, Fas ligand (FasL), inducible nitric oxide sythase and nitrotyrosine determined by immunohistochemical analysis were reduced markedly in lung tissues from TNF-alphaR1KO at 4 h and 24 h after carrageenan injection. Moreover, TNF-alpha and interleukin-1beta concentrations were reduced in inflamed areas and in pleural exudates from TNF-alphaR1KO. To support the results generated using pleural inflammation, carrageenan-induced paw oedema models were also performed. In order to elucidate whether the observed anti-inflammatory effects were related to the inhibition of TNF-alpha, we also investigated the effect of etanercept, a TNF-alpha soluble receptor construct, on carrageenan-induced pleurisy. The treatment with etanercept (5 mg/kg subcutaneously 2 h before the carrageenan injection) reduces markedly both laboratory and histological signs of carrageenan-induced pleurisy. Our results showed that administration of etanercept resulted in the same outcome as that of deletion of the TNF-alphaR1 receptor, adding a new insight to TNF-alpha as an excellent target by therapeutic applications.

Fig. 1

Effects of tumour necrosis factor (TNF)-α gene deletion and etanercept administration on CAR-induced pleural exudate production (a) and accumulation of polymorphonuclear cells in the pleural cavity (b). At 4 h (polymorphonuclear leucocytes are the predominant cell type) and 24 h (peak of total inflammatory cell number and exudate volume), the total volume was recovered by aspiration. A significant production in pleural exudate (a) and polymorphonuclear cell infiltration (b) was observed in the pleural cavity from wild-type (WT) mice at 4 h and 24 h after CAR administration. The absence of TNF-α receptor 1 gene in mice as well as the treatment of WT mice with etanercept reduced significantly the presence of pleural exudate (a) and the number of inflammatory cells in the pleural cavity (b) at 4 h and 24 h after CAR injection. Data are means ± standard error of the mean of 10 mice for each group. *P < 0·01 versus sham; °P < 0·01 versus CAR-WT group. KO, knock-out; CAR, carrageenan.

Fig. 2

Effects of tumour necrosis factor (TNF)-αR1 gene deletion and etanercept administration on CAR-induced neutrophils infiltration in lung tissues. Myeloperoxidase (MPO) activity was elevated significantly at 4 h and 24 h after CAR administration in wild-type (WT) mice. In TNF-α receptor 1 KO mice lung MPO activity was reduced significantly at 4 h and 24 h. Similarly, the treatment of WT mice with etanercept (5 mg/kg administered subcutaneously 2 h prior to CAR) reduced significantly neutrophil infiltration in the lung tissues at 4 h and at 24 h after CAR administration. Data are means ± standard error of the mean of 10 mice for each group. *P < 0·01 versus sham; °P < 0·01 versus CAR-WT group. KO, knock-out; CAR, carrageenan.

Fig. 3

Effects of tumour necrosis factor (TNF)-α gene deletion and etanercept administration on lung injury. Lung tissues collected at 4 h and 24 h were stained with haematoxylin and eosin. Histological examination of lung sections collected at 4 h from all wild-type (WT) carrageenan-injected mice showed tissue injury as well as inflammatory cells infiltration (c). At 24 h after carrageenan administration a significant augmentation of lung injury as well as a significant presence in inflammatory cells (d) was observed in the lung tissues collected from WT mice. The absence of TNF-α receptor 1 (TNF-αR1) in mice in TNF-αR1 knock-out mice led to a significant reduction in lung injury and inflammatory cells infiltration at 4 h (e) and 24 h after carrageenan administration (g). Similarly, the treatment of WT mice with etanercept (5 mg/kg administered subcutaneously 2 h prior carrageenan) reduced significantly at 4 h (f) and 24 h (h) after carrageenan injection of the lung injury. No injury was observed in the lung tissues collected from sham WT mice (a) and from sham TNF-αR1KO mice (b). Figure is representative of at least three experiments performed on different experimental days.

Fig. 4

Effects of tumour necrosis factor-α receptor 1 (TNF-αR1) gene deletion and etanercept administration on lung TNF-α and interleukin (IL)-1β levels. TNF-α and IL-1β production was evaluated in the pleural exudate and lung tissues collected at 4 h and 24 h after CAR injection using an enzyme-linked immunosorbent assay kit. A significant production TNF-α (a) and IL-1β (b) was observed in pleural exudate collected from wild-type (WT) mice. The absence of TNF-αR1 gene in mice as well as the treatment of WT mice with etanercept reduced significantly the pleural exudate production of TNF-α (a) and IL-1β (b). Similarly, a significant increase in TNF-α (c) and IL-1β (d) was observed in the lung tissues from CAR-injected WT mice at 4 h and 24 h after CAR. In the lung tissues from CAR-injected TNF-αR1KO mice as well as of WT mice which received etanercept TNF-α (c) and IL-1β (d) levels were reduced significantly in comparison with those of WT animals. Data are means ± standard error of the mean of 10 mice for each group. *P < 0·01 versus SHAM; °P < 0·01 versus CAR-WT group. KO, knock-out; CAR, carrageenan.

Fig. 5

Immunohistochemical localization of tumour necrosis factor (TNF)-α in the lung. Lung sections were taken 4 h after injection of carrageenan. No positive staining for TNF-α was observed in the lung tissues collected from sham wild-type (WT) mice (a) and from sham TNF-α receptor 1 knock-out (TNF-αR1KO) mice (b). In contrast, positive staining for TNF-α (c), localized mainly in the infiltrated inflammatory cells and pneumocytes as well as in vascular wall (see arrows), was observed in lung sections from WT animals. In carrageenan-injected TNF-αR1KO mice, no positive staining for TNF-α was observed in the lung tissues (d). Similarly, the treatment of WT mice with etanercept (5 mg/kg administered subcutaneously 2 h prior to carrageenan) reduced positive staining visibly and significantly for TNF-α in the lung tissues (e). Figure is representative of at least three experiments performed on different experimental days.

Fig. 6

Immunohistochemical localization of interleukin (IL)-1β in the lung. Lung sections shown were taken 4 h after injection of carrageenan. No positive staining for IL-1β was observed in the lung tissues collected from sham wild-type (WT) mice (a) and from sham tumour necrosis factor-α receptor 1 knock-out (TNF-αR1KO) mice (b). At 4 h after carrageenan administration, positive staining for IL-1β localized in the infiltrated inflammatory cells and pneumocytes as well as in vascular wall (c, see arrows) was observed in lung tissue sections obtained from WT animals. The absence of a functional TNF-αR1 gene resulted in a significant reduction of the positive staining for IL-1β (d) in the infiltrated inflammatory cells and pneumocytes as well as in vascular wall (e). Similarly, treatment of WT mice with etanercept (5 mg/kg administered subcutaneously 2 h prior to carrageenan) reduced positive staining visibly and significantly for IL-1β in the lung tissues (e). Figure is representative of at least three experiments performed on different experimental days.

Fig. 7

Typical densitometry evaluation. Densitometry analysis of immunocytochemistry photographs (n = 5) for intercellular adhesion molecule 1 (ICAM-1), tumour necrosis factor (TNF)-α, interleukin-1 (IL-1), P-selectin, inducible nitric oxide sythase (NOS), nitrotyrosine and Fas ligand (FasL) from lung was assessed. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). Data are expressed as percentage of total tissue area; n.d., not detectable. *P < 0·01 versus sham. °P < 0·01 versus carrageenan (CAR). WT, wild-type; KO, knock-out.

Fig. 8

Immunohistochemical localization of intercellular adhesion molecule 1 (ICAM-1) in the lung. Lung tissues shown were taken 4 h after injection of carrageenan. No positive staining for ICAM-1 was found in lung tissue section from sham wild-type (WT) mice (a) and sham tumour necrosis factor-α receptor 1 knock-out (TNF-αR1KO) mice (b). In contrast, an increase in imununohistochemical staining for ICAM-1 was observed in lung tissue sections obtained from WT mice (c) localized mainly around the vessels (see arrows). In carrageenan-treated TNF-αR1KO mice, the positive immunostaining in the lung for ICAM-1 was reduced visibly and significantly (d). Similarly, the treatment of TNF-α-R1 WT mice with etanercept (5 mg/kg administered subcutaneously 2 h prior to carrageenan) reduced the positive immunostaining for ICAM-1 in the lung tissues (e). Figure is representative of at least three experiments performed on different experimental days.

Fig. 9

Immunohistochemical localization of P-selectin in the lung. Lung tissues shown were taken 4 h after injection of carrageenan. No positive staining for P-selectin was found in lung tissue sections from sham wild-type (WT) mice (a) and from sham tumour necrosis factor-α receptor 1 knock-out (TNF-αR1KO) mice (b). In contrast, an increase in imununohistochemical staining for P-selectin was observed in lung tissue sections obtained from WT mice (c) localized mainly around the vessels (see arrows). In carrageenan-treated TNF-αR1KO mice, the positive immunostaining for P-selectin was reduced visibly and significantly (d). Similarly, the treatment of TNF-α-R1 WT mice with etanercept (5 mg/kg administered subcutaneously 2 h prior to carrageeenan) reduced the positive immunostaining for P-selectin in the lung tissues (e). Figure is representative of at least three experiments performed on different experimental days.

Fig. 10

Effects of tumour necrosis factor-α receptor 1 (TNF-αR1) gene deletion and etanercept administration on inducible nitric oxide synthase (iNOS) expression and nitrite/nitrate formation. iNOS expression was studied by immunohistochemical localization in the lung collected at 4 h after injection of carrageenan. No positive staining for iNOS was observed in the lung tissues obtained from sham wild-type (WT) mice (a) and sham TNF-αR1 knock-out (KO) mice (b). In contrast, tissue sections obtained from WT animals at 4 h after carrageenan injection demonstrate positive staining for iNOS localized mainly in the infiltrated inflammatory cells (c, see arrows). In carrageenan-injected TNF-αR1KO mice, no positive staining for iNOS were observed in the lung tissues (d). Similarly, the treatment of WT mice with etanercept (5 mg/kg administered subcutaneously 2 h prior to carrageenan) reduced positive staining visibly and significantly for iNOS in the infiltrated inflammatory cells in the lung tissues (e). Nitrite/nitrate levels were also increased significantly in the exudate obtained from WT mice at 4 h and 24 h after carrageenan injection (f). Nitric oxide exudate levels were reduced significantly in carrageenan-injected TNF-αR1KO mice as well as in WT mice treated with etanercept (5 mg/kg administered subcutaneously 2 h prior to carrageenan) (f). Figure is representative of at least three experiments performed on different experimental days. Data are means ± standard error of the mean of 10 mice for each group. *P < 0·01 versus sham; °P < 0·01 versus carrageenan-WT group.

Fig. 11

Effects of tumour necrosis factor-α receptor 1 (TNF-αR1) gene deletion and etanercept administration on nitrotyrosine formation and lipid peroxidation. Nitrotyrosine formation was evaluated by immunohistochemical localization in the lungs collected at 4 h after injection of carrageenan. No positive staining for nitrotyrosine was observed in the lung tissues obtained from sham wild-type (WT) mice (a) and sham TNF-αR1 knock-out (KO) mice (b). In contrast, tissue sections obtained from WT animals at 4 h after carrageenan administration demonstrate positive staining for nitrotyrosine localized mainly in the infiltrated inflammatory cells (c, see arrows). In carrageenan-treated TNF-αR1KO mice, no positive staining for nitrotyrosine was observed in the lung tissues (d). Similarly, the treatment of WT mice with etanercept (5 mg/kg administered intraperitoneally 2 h prior to carrageenan) reduced positive staining visibly and significantly for nitrotyrosine in the infiltrated inflammatory cells in the lung tissues (e). In addition, at 4 h and 24 h after carrageenan-induced pleurisy, malondialdehyde bis (dimethyl acetal) 99% levels were increased significantly in the lungs from carrageenan-treated mice at 4 h and 24 h after carrageenan administration (f). Lipid peroxidation was attenuated significantly in carrageenan- treated TNF-αR1KO mice as well as in WT mice treated with etanercept (5 mg/kg administered subcutaneously 2 h prior to carrageenan) (f). Figure is representative of at least three experiments performed on different experimental days. Data are means ± standard error of the mean of 10 mice for each group. *P < 0·01 versus sham; °P < 0·01 versus carrageenan-WT group.

Fig. 12

Immunohistochemical localization of Fas ligand (FasL) in the lung. Lung tissues were taken 4 h after injection of carrageenan. No positive staining for FasL was observed in the lung tissues from sham wild-type (WT) mice (a) and sham tumour necrosis factor-α receptor 1 knock-out (TNF-αR1KO) mice (b). Positive staining for FasL (c) localized mainly in vascular wall and in pneumocytes (see arrows) were detected in the lung tissues from WT mice. The absence of a functional TNF-αR1 gene in TNF-αR1KO mice resulted in a significant reduction of the positive staining for FasL (d) in the infiltrated inflammatory cells and pneumocytes as well as in vascular wall (e). Similarly, the treatment of WT mice with etanercept (5 mg/kg administered subcutaneously 2 h prior to carrageenan) reduced positive staining for FasL visibly and significantly in the lung tissues (e). Figure is representative of at least three experiments performed on different experimental days.

Fig. 13

Effects of tumour necrosis factor-α receptor 1 (TNF-αR1) gene deletion and etanercept administration on paw oedema development (a) and neuthophil infiltration (b) induced by carrageenan subplantar injection in the mice. The paw volume was measured before the subplantar injection and at 1-h intervals up to 5 h. The oedema volume is the difference in the paw volume at each time-point and the basal paw volume. Myeloperoxidase (MPO) activity was increased significantly in the paw from wild-type (WT) mice collected at 4 h after carrageenan (b). The absence of TNF-αR1 gene in knock-out mice (TNF-αR1KO) as well as the treatment of WT mice with etanercept reduced significantly the carrageenan-induced paw oedema development at all time-points (a). Moreover, MPO activity was reduced in significantly carrageenan-treated TNF-αR1KO mice as well as in WT mice pretreated with etanercept (b). Data are means ± standard error of the mean of 10 mice for each group. #P < 0·01 versus carrageenan-WT group at the indicated time-points. *P < 0·01 versus SHAM; °P < 0·01 versus carrageenan-WT group.