THE EFFECT OF TNFα ON THE PRODUCTION OF NITRIC OXIDE BY NEUTROPHILS ISOLATED FROM HEIFERS IN THE COURSE OF BOVINE RESPIRATORY DISEASE

Joanna Wessely-Szponder
Department of Pathophysiology, Faculty of Veterinary Medicine, Agricultural University in Lublin, Poland

ABSTRACT

In the course of Bovine Respiratory Disease (BRD), lung injury is caused, among others, by neutrophil products, including nitric oxide (NO). Destructive action of this free radical on respiratory tract tissues is stimulated by some cytokines. The purpose of this study was to determine how TNFα influences on NO generation by neutrophils in the course of BRD. This study revealed that neutrophils isolated from heifers with acute BRD produced greater amounts of NO than in chronic BRD. In all phases of BRD and in both forms of the disease the concentration of TNFα in the range of 0.05 ng·ml^-1 to 0.5 ng·ml^-1 had a stimulatory effect on neutrophils after 0.5 h and 24 h incubation. Moreover, the greatest NO production was observed in cultures of neutrophils from an acute BRD in the concentration of 0.5 ng·ml^-1 after 0.5 h and 24h incubation. After 48h incubation NO production increased only at a concentration of 0.05 ng·ml^-1. Above these concentrations production of NO decreased. The most pronounced inhibitory action of TNFα on generation of NO by neutrophils became in acute BRD in cultures after 0.5 h at 50 ng·ml^-1, and after 24 h at 5 ng·ml^-1, and was related to diminished viability of neutrophils.

Key words: Bovine Respiratory Disease, TNFα, nitric oxide, neutrophil.

INTRODUCTION

Bovine respiratory disease (BRD) is a significant economical problem in bovine farms and neutrophil constituents lead to lung injury and worsening the course of the disease [15,16,19]. Apart from many other products, such as elastase, myeloperoxidase (MPO), and alkaline phosphatase (ALKP), neutrophils also generate free radicals, such as nitric oxide (NO)[1,6,13,18,19,20]. This free radical, generated by neutrophils during inflammation, modulates both acute and chronic inflammatory reactions, moreover the number of studies have shown that NO is involved in the tissue damage by initiating lipid peroxidation, DNA oxidation, or inactivation of enzymes and proteins especially in the airway inflammation [2,8]. Moreover superoxide anion, rapidly reacting with NO, yields peroxynitrite. The release of this compound may be a strong weapon against invading microorganisms, but overproduction or uncontrolled formation of peroxynitrite is an important factor in the tissue destruction during inflammatory process [3,4,13,14]. The release of NO by inflammatory cells such as neutrophils can be regulated by several cytokines [18]. However, little is known about the role of NO in the lung injury in the course of BRD and the participation of proinflammatory cytokines in this process. The aim of this study was to evaluate the influence of TNFα, a major cytokine in inflammatory response, on generation of NO by neutrophils isolated from heifers in the course of acute and chronic BRD.

MATERIAL AND METHODS

The study was carried out on 90 heifers. Clinical examination of each heifer was performed before collection of blood. On the basis of clinical signs and duration of disease heifers were divided into two groups: acute and chronic BRD. The third group consisted of healthy heifers. Peripheral blood was collected from three groups of heifers: 30 animals with acute BRD (three times in 4 day intervals), 30 with chronic BRD (in 14 day intervals), and from 30 healthy heifers (n=15 in 4 day intervals, and n=15 in 14 day intervals). These periods correspond to three phases of disease. Neutrophils were isolated according to the method of Mottola [5]. The remaining pellet was washed with phosphate-buffered saline (PBS) and the final cell pellet was resuspended in 1 ml of Dulbecco’s Modified Eagle’s Medium (DMEM-Sigma). After isolation, viability of PMNs cells was determined by trypan blue exclusion. After cells counting and differentiation cell suspensions were adjusted to a final concentration of 2 x 10⁶ cells/ml. Then, cell cultures were incubated at 37°C and 5% CO₂ with 0, 0.05, 0.5, 5 and 50 ng·ml^-1 of human recombinant TNFα, control groups were supplemented by PBS in equal volume. Nitric oxide level was measured after 0.5, 24, 48 and 72 hours of incubation as well as neutrophil viability. Nitric oxide level was determined by Griess reaction: 50 μl of supernatant were mixed with 200 μl of Griess reagent (1% sulfanilamide, 0.1% naphthylendiamine dihydrochloride and 2.5% H₃PO₄). Absorbance at 545 nm was measured after 10 min. incubation with Griess reagent and compared with a standard. Obtained values were expressed as a concentration of nitrite, the stable product of NO, which accumulates in medium [7,9,10,11,12]. Examined values were compared using analysis of variance and Student’s t-test and differences were considered as significant at p<0.05.

RESULTS

Neutrophils isolated from heifers with acute BRD produced NO at the level of 3.0±0.57 μM nitrite after 0.5 h of incubation, and 6.2±0.8 μM nitrite after 72 h. In the course of chronic BRD values obtained were lower than in the acute BRD (2.12±0.3 μM nitrite and 4.5±0.3 μM nitrite, respectively). The PMN obtained from healthy heifers in the same time generated lower amounts of NO (from 1.24±0.44 μM nitrite after 0.5 h to maximum after 72 h incubation)(Fig. 1). In the second phase of the disease value of generated NO was lower, and this level increased during 72 h incubation to the smaller degree. In the third phase generation of NO approximated to values obtained from the healthy heifers. In all phases and in both forms of BRD the concentration of TNFα of 0.05 ng·ml^-1 and 0.5 ng·ml^-1 had stimulatory effect on neutrophils after 0.5 h and 24 h incubation. The greatest response was observed in the concentration of 0.5 ng·ml^-1 after 0.5 h and 24 h incubation (Fig. 2). After 48 h incubation the augmentation of NO production was only in the concentration of 0.05 ng·ml^-1. In contrast, in cultures from healthy heifers increase of NO production was noted only in the presence of 0.05 ng·ml^-1 TNFα.(Fig. 2). The treatment of neutrophils from healthy heifers with TNFα in the concentrations from 0.5 to 50 ng·ml^-1 resulted in inhibition of NO generation that started after 0.5 h of incubation (Fig. 3). In an acute BRD, in turn, inhibitory action of TNFα on generation of NO by neutrophils was observed after 0.5 h in 50 ng·ml^-1, and after 24 h in 5 ng·ml^-1, and was correlated with diminishing viability of neutrophils (Fig. 3).

DISCUSSION

The results of our study revealed that generation of nitric oxide by neutrophils depended on a concentration of TNFα, time of incubation and clinical phase of BRD (chronic or acute). Only concentration of 0.05 ng·ml^-1 of TNFα activates generation of NO by neutrophils of healthy heifers. Whereas neutrophils from BRD heifers are activated by TNFα in the concentration from 0.05 to 0.5 ng·ml^-1. Augmented production of NO by neutrophils from heifers with BRD may lead to lung injury and worsening the course of disease [16,20].

There are some discrepancies between authors about the influence of TNFα on NO generation by neutrophils. Misso et al. [7] observed, that human neutrophils treated by 6 to 16 h with 500 U/ml TNFα inhibited production of NO, which was explained by apoptosis appeared after 6 h. Inhibitory effect of high concentrations of TNFα in cultures of bovine neutrophils in present study may be also explained by this effect, more pronounced in group of neutrophils from acute BRD heifers. According to other authors TNFα by activation iNOS increases the generation of NO by human neutrophils [10]. The same effect was observed in present experiment during initial activation of bovine neutrophils together with increase of NO level. According to Roy et al. [12] NO produced by neutrophils in respiratory disorders generated lung injury. They pointed out that stimulation of buffalo’s neutrophils by LPS from P. multocida caused increase of NO production by these cells. Released NO reacting with O₂ and yielded peroxynitrite, with potential for lung tissue injury. This very reactive product is responsible for lipid peroxidation, leading to endothelial cell destruction in the course of respiratory infection in cattle, similarly like in ARDS [3,16]. The influence of infectious agent on production of NO by neutrophils stimulated with TNFα was investigated by Tsukahara et al. [17]. They revealed that simultaneous stimulation of human neutrophils by TNFα and LPS causes both expresion of mRNA iNOS and release NO to culture medium after 6 h incubation. In the course of infection such as sepsis or endotoxaemia both mRNA expresion for iNOS and generation of NO increases, which is caused by bacterial agents as well as endogenous host mediators [17]. This experiment revealed similar effect in heifers during BRD, when stronger response of neutrophils should be explained by neutrophil stimulation with bacterial agents. In the case of acute BRD neutrophil response was more intense because of stronger stimulation of neutrophils by bacterial agents than in case of chronic disease.

CONCLUSIONS

1. The generation of nitric oxide by neutrophils depended on concentration of TNFα, time of incubation, and the group of animals.

2. The greatest NO production was observed in cultures of neutrophils from the acute BRD in the concentration of 0.5 ng·ml^-1 after 0.5 h and 24 h incubation.

3. Augmented production of NO by neutrophils from heifers with BRD may lead to lung injury and worsening the course of disease.

REFERENCES

1. Abu-Sound H., Hazen S., 2000. Nitric oxide is a physiological substrate for mammalian peroxidases. J. Biol. Chem. 275, 37524-37532.

2. Armstrong R., 2001. The physiological role and pharmacological potential of nitric oxide in neutrophil activation. Int. Immunopharmacol. 1, 1501-1512.

3. Dőrger M., Allmeling A-M., Kiefmann R., Schropp A., Krombach F., 2002. Dual role of inductible nitric oxide synthase in acute asbestos-induced lung injury. Free Rad. Biol. Med. 33, 491-501.

4. Goff W.L., Johnson W.C., Wyatt C.R., Cluff C.W., 1996. Assessment of bovine mononuclear phagocytes and neutrophils for induced L-arginine-derended nitric oxide production. Vet. Immunol. Immunopathol. 55, 45-62.

5. Hoeben D., Dosogne H., Heyneman R., Burvenich C., 1997. Effect of antibiotics on the phagocytic and respiratory burst activity of bovine granulocytes. Eur. J. Pharm. 332, 289-297.

6. Ishida-Okawara A., Tsuchiya T., Nunoi H., Mizuno S., Suzuki K., 1996. Modulation of degranulation and superoxide generation in human neutrophils by unsaturated fatty acids of odd carbon numers. Biochim. Biophys. Acta 1314, 239-246.

7. Misso N., Peacock C.D., Watkins D.N., Thompson P.J., 2000. Nitrite generation and antioxidant effects during neutrophil apoptosis Free Rad. Biol. Med. 28, 934-943.

8. Muijsers R., Folkerts G., Henricks P., Sadeghi-Hashjin G., Nijkamp F., 1997. Peroxynitrite: A two-faced metabolite of nitric oxide. Life Sci. 60, 1833-1845.

9. Nims R., Darbyshire J.F., Saavedra J.E., Christodoulou D., Hanbauer I., Cox G.W., Grisham M., Krishna M., Wink D., 1995. Colorimetric methods for the determination of nitric oxide concentration in neutral aqueous solutions. Comp. Meth. Enzymol. 7, 48-54.

10. Ridnour L., Sim J., Hayward M., Wink D., Martin S., Buetter G., Spitz R., 2000. A spectrophotometric method for the direct detection and quantitation of nitric oxide, nitrite, and nitrate in cell culture media. Analytical Biochemistry 281, 223-229.

11. Robbins R., Grisham M., 1997. Nitric oxide. Int. J. Biochem. Cell Biol. 29, 857-860.

12. Roy S.C., More T., Pati U.S., Srivastava S.K., 1996. Effect of P. multocida vaccination on buffalo polymorphonuclear hydrogen peroxide and nitric oxide production Vet. Immunol. Immunopathol. 51, 173-178.

13. Ródenas J., Mitjavila M., Carbonell T., 1995. Simultaneous generation of nitric oxide and superoxide by inflammatory cells in rats. Free Rad. Biol. Med. 18, 869-875.

14. Seti S., Dikshit M., 2000. Modulation of polymorphonuclear leukocytes function by nitric oxide. Thromb. Res. 100, 223-247.

15. Stockley R., 1995. Role of inflammation in respiratory tract infections. Am. J. Med. 29, 8-10.

16. Tavaf-Motamed H., Miner T.J., Starnes B.W., Shea-Donohue T., 1998. Nitric oxide mediates acute lung injury by modulation of inflammation. J. Surg. Res. 78, 137-142.

17. Tsukahara Y., Morisaki T., Kojima M., Uchiyama A., Tanaka M., 2001. iNOS expression by activated neutrophils from patients with sepsis. ANZ J. Surg. 71, 15-20.

18. Wessely-Szponder J., 2006. Udział cytokin w destrukcyjnej odpowiedzi neutrofili w przebiegu zespołu oddechowego u jałówek [Participation of cytokines in destructive response of neutrophils during BRD in heifers]. Doctor thesis, Department of Pathophysiology, Chair of Preclinical Veterinary Sciences, Agricultural Academy, Lublin [in Polish].

19. Wessely-Szponder J., Bobowiec R., 2005. Znaczenie czynników endogennych, w tym neutrofili, w patogenezie stanów zapalnych układu oddechowego u bydła [The role of endogenic factors, including neutrophils, in the pathogenesis of bovine respiratory tract infection]. Życie Weterynaryjne 5, 274-278 [in Polish].

20. Wessely-Szponder J., Bobowiec R., Martelli F., Wójcik M., Kosior-Korzecka U., 2004. Assessment of neutrophil components as markers of lung injury in the course of bovine respiratory tract infection. Polish J. Vet. Sci. 7, 157-161.

Accepted for print: 3.09.2007

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