Anat Cell Biol.  2010 Dec;43(4):294-302. 10.5115/acb.2010.43.4.294.

Mega-dose vitamin C attenuated lung inflammation in mouse asthma model

• Jeong YJ
• Kim JH
• Kang JS
• Lee WJ
• Hwang YI

• KMID: 1455343
• DOI: http://doi.org/10.5115/acb.2010.43.4.294

Abstract

Asthma is a Th2-dependent disease mediated by IgE and Th2 cytokines, and asthmatic patients suffer from oxidative stresses from abnormal airway inflammation. Vitamin C is a micro-nutrient functioning as an antioxidant. When administered at a mega-dose, vitamin C has been reported to shift immune responses toward Th1. Thus, we tried to determine whether vitamin C exerted beneficial effects in asthma animal model. Asthma was induced in mice by sensitizing and challenging with ovalbumin. At the time of challenge, 3~5 mg of vitamin C was administered and the effects were evaluated. Vitamin C did not modulate Th1/Th2 balance in asthma model. However, it decreased airway hyperreactivity to methacholine, decreased inflammatory cell numbers in brochoalveolar lavage fluid, and moderate reduction of perivascular and peribronchiolar inflammatory cell infiltration. These results suggest that vitamin C administered at the time of antigen challenge exerted anti-inflammatory effects. Further studies based on chronic asthma model are needed to evaluate a long-term effect of vitamin C in asthma. In conclusion, even though vitamin C did not show any Th1/Th2 shifting effects in this experiment, it still exerted moderate anti-inflammatory effects. Considering other beneficial effects and inexpensiveness of vitamin C, mega-dose usage of vitamin C could be a potential supplementary modality for the management of asthma.

Keyword

Asthma; Vitamin C; Lung inflammation; Th1/Th2 balance

MeSH Terms

Animals
Ascorbic Acid
Asthma
Cell Count
Cytokines
Humans
Immunoglobulin E
Inflammation
Lung
Methacholine Chloride
Mice
Models, Animal
Ovalbumin
Oxidative Stress
Pneumonia
Therapeutic Irrigation
Vitamins
Ascorbic Acid
Cytokines
Immunoglobulin E
Methacholine Chloride
Ovalbumin
Vitamins

Figure

• 

  Fig. 1 Plethysmography of normal and asthma-induced mice. Mice were inhaled with aerosol containing methacholine at concentrations indicated and placed in the plethymography chamber. Breathing was recorded for 2 min and airway resistance was calculated thereby. PBS-treated mice with asthma (■) showed increased airway resistance compared to normal mice (◆) at all concentrations of methacholine. Meanwhile, vitamin C-treated mice with asthma (▲) showed decreased resistance than that of PBS-injected mice, and that was similar to that of normal mice at methacholline concentrations from 6.25 to 25 mg/ml. *P<0.05.

• 

  Fig. 2 (left panel) Cell numbers in BALF. Mice were anesthesized and 1 ml of saline was infused into the lung through a needle in the trachea. The fluid was re-drawn after 1 min and centrifuged. Obtained cells were re-suspended, and cell count was done. Scanty cells were found in BALF from normal mice while many inflammatory cells were observed in BALF from asthma-induce mice. Vitamin C-treated mice showed less number of BALF cells than PBS-injected mice. A representative profile of three independent experiments. n=8 for each group. *P<0.05. (right panel) Differential cell counts of BALF cells from asthmatic mice. Cells were spread on a glass slide, stained with Wright's solution, and differentially counted under light microscope. In both experimental groups, eosinophils comprised about two thirds of total cells, and the other one third was almost macrophages. Lymphocytes and neutrophils were observerd with a very low frequency. These two groups showed no difference in cellular composition of BALF (P>0.05). Over 1,000 cells were counted in each group.

• 

  Fig. 3 Wright staining of BALF cells. After centrifugation of BALF, sedimented cells were re-suspended in 300 µl PBS containing 0.05% albumin and monolayer cell spread was prepared by cytospin. In normal mice, cells were scanty and almost all cells were macrophages with a quiescent appearance (left panels). Cells from mice with asthma, irrespective of PBS (middle panels) or vitamin C treatment (right panels), were abundant in number and mostly composed of eosinophils and macrophages. Macrophages in these groups differed from those of normal mice in their appearance and size. ×100 magnification (upper panels) and ×800 magnification (lower panels).

• 

  Fig. 4 Histological scoring of lung tissue. Paraffin sections were stained with hematoxylin and eosin (A~F) or with periodic acid-Schiff's reagent (G~I). The intensity of perivascular inflammatory cell infiltration (upper panels) or of peribronchiolr infiltration (middle panels) were semi-quantified as the numbers of cell layers and were depicted in graphs on the right column. Mucus hyperplasia were quantified as the ratio that goblet cells occupied on the respiratory epithelium and also depicted in the graph on the right column. Vitamin C administration significantly decreased the perivascular cell infiltration and tended to decrease peribronchiloar cell infiltration. However, the intensity of mucus hyperplasia was not affected by vitamin C treatment.

• 

  Fig. 5 OVA-specific serum antibody titers after asthma induction. Mice were sensitized on days 1, 2, and 3 by peritoneal injection of 100 µg ovalbumin immersed in alum and challenged on days 18, 19, 22, and 23, by intranasal dripping of 25 µg OVA/30 µl PBS. On day 25, blood samples were drawn from orbital venous plexus. Sera were obtained and OVA-specific ELISA titrations were performed for each isotype. Both the PBS-injected and vitamin C-treated mice showed elevated levels of antigen-specific titers in all isotypes. However, there was no statistically significant differences in the titers between these two groups. A representative profile of three independent experiments. n=8 for each group.

Reference

1. Barrios RJ, Kheradmand F, Batts L, Corry DB. Asthma: pathology and pathophysiology. Arch Pathol Lab Med. 2006. 130:447–451.

2. Bjermer L, Diamant Z. Current and emerging nonsteroidal anti-inflammatory therapies targeting specific mechanisms in asthma and allergy. Treat Respir Med. 2004. 3:235–246.

3. Broide DH. Immunologic and inflammatory mechanisms that drive asthma progression to remodeling. J Allergy Clin Immunol. 2008. 121:560–570.

4. Cárcamo JM, Pedraza A, Bórquez-Ojeda O, Golde DW. Vitamin C suppresses TNF alpha-induced NF kappa B activation by inhibiting I kappa B alpha phosphorylation. Biochemistry. 2002. 41:12995–13002.

5. Chanez P, Wenzel SE, Anderson GP, et al. Severe asthma in adults: what are the important questions? J Allergy Clin Immunol. 2007. 119:1337–1348.

6. Chang HH, Chen CS, Lin JY. High dose vitamin C supplementation increases the Th1/Th2 cytokine secretion ratio, but decreases eosinophilic infiltration in bronchoalveolar lavage fluid of ovalbumin-sensitized and challenged mice. J Agric Food Chem. 2009. 57:10471–10476.

7. Cohn L, Tepper JS, Bottomly K. IL-4-independent induction of airway hyperresponsiveness by Th2, but not Th1, cells. J Immunol. 1998. 161:3813–3816.

8. Desai D, Brightling C. Cytokine and anti-cytokine therapy in asthma: ready for the clinic? Clin Exp Immunol. 2009. 158:10–19.

9. Doherty T, Broide D. Cytokines and growth factors in airway remodeling in asthma. Curr Opin Immunol. 2007. 19:676–680.

10. Du WD, Yuan ZR, Sun J, et al. Therapeutic efficacy of high-dose vitamin C on acute pancreatitis and its potential mechanisms. World J Gastroenterol. 2003. 9:2565–2569.

11. Duan W, Chan JH, Wong CH, Leung BP, Wong WS. Anti-inflammatory effects of mitogen-activated protein kinase kinase inhibitor U0126 in an asthma mouse model. J Immunol. 2004. 172:7053–7059.

12. Fogarty A, Lewis SA, Scrivener SL, et al. Oral magnesium and vitamin C supplements in asthma: a parallel group randomized placebo-controlled trial. Clin Exp Allergy. 2003. 33:1355–1359.

13. Fogarty A, Lewis SA, Scrivener SL, et al. Corticosteroid sparing effects of vitamin C and magnesium in asthma: a randomised trial. Respir Med. 2006. 100:174–179.

14. Gao J, Gao X, Li W, Zhu Y, Thompson PJ. Observational studies on the effect of dietary antioxidants on asthma: a meta-analysis. Respirology. 2008. 13:528–536.

15. Guaiquil VH, Vera JC, Golde DW. Mechanism of vitamin C inhibition of cell death induced by oxidative stress in glutathione-depleted HL-60 cells. J Biol Chem. 2001. 276:40955–40961.

16. Haines DD, Varga B, Bak I, et al. Summative interaction between astaxanthin, Ginkgo biloba extract (EGb761) and vitamin C in Suppression of respiratory inflammation: a comparison with ibuprofen. Phytother Res. 2010. [Epub ahead of Print].

17. Hamid Q, Tulic M. Immunobiology of asthma. Annu Rev Physiol. 2009. 71:489–507.

18. Harik-Khan RI, Muller DC, Wise RA. Serum vitamin levels and the risk of asthma in children. Am J Epidemiol. 2004. 159:351–357.

19. Hernandez M, Zhou H, Zhou B, et al. Combination treatment with high-dose vitamin C and alpha-tocopherol does not enhance respiratory-tract lining fluid vitamin C levels in asthmatics. Inhal Toxicol. 2009. 21:173–181.

20. Holgate ST, Holloway J, Wilson S, et al. Understanding the pathophysiology of severe asthma to generate new therapeutic opportunities. J Allergy Clin Immunol. 2006. 117:496–506.

21. Izuhara K, Ohta S, Shiraishi H, et al. The mechanism of mucus production in bronchial asthma. Curr Med Chem. 2009. 16:2867–2875.

22. Kamgar M, Zaldivar F, Vaziri ND, Pahl MV. Antioxidant therapy does not ameliorate oxidative stress and inflammation in patients with end-stage renal disease. J Natl Med Assoc. 2009. 101:336–344.

23. Kaur B, Rowe BH, Arnold E. Vitamin C supplementation for asthma. Cochrane Database Syst Rev. 2009. (1):CD000993.

24. Kelly FJ, Mudway I, Blomberg A, Frew A, Sandström T. Altered lung antioxidant status in patients with mild asthma. Lancet. 1999. 354:482–483.

25. Kirby AC, Yrlid U, Svensson M, Wick MJ. Differential involvement of dendritic cell subsets during acute Salmonella infection. J Immunol. 2001. 166:6802–6811.

26. Langlois M, Duprez D, Delanghe J, De Buyzere M, Clement DL. Serum vitamin C concentration is low in peripheral arterial disease and is associated with inflammation and severity of atherosclerosis. Circulation. 2001. 103:1863–1868.

27. Lee CW, Wang XD, Chien KL, et al. Vitamin C supplementation does not protect L-gulono-gamma-lactone oxidase-deficient mice from Helicobacter pylori-induced gastritis and gastric premalignancy. Int J Cancer. 2008. 122:1068–1076.

28. Maeda N, Hagihara H, Nakata Y, Hiller S, Wilder J, Reddick R. Aortic wall damage in mice unable to synthesize ascorbic acid. Proc Natl Acad Sci U S A. 2000. 97:841–846.

29. Marleau AM, Summers KL, Singh B. Differential contributions of APC subsets to T cell activation in nonobese diabetic mice. J Immunol. 2008. 180:5235–5249.

30. McMillan SJ, Lloyd CM. Prolonged allergen challenge in mice leads to persistent airway remodelling. Clin Exp Allergy. 2004. 34:497–507.

31. Naidu KA. Vitamin C in human health and disease is still a mystery? An overview. Nutr J. 2003. 2:7.

32. Nelde A, Teufel M, Hahn C, et al. The impact of the route and frequency of antigen exposure on the IgE response in allergy. Int Arch Allergy Immunol. 2001. 124:461–469.

33. Noh K, Kim HG, Shin YA, et al. The effects of high-dose vitamin C administration on the cell-mediated immune response in mice. Immune Netw. 2003. 3:211–218.

34. Noh K, Lim H, Moon SK, et al. Mega-dose Vitamin C modulates T cell functions in Balb/c mice only when administered during T cell activation. Immunol Lett. 2005. 98:63–72.

35. Rizzo MR, Abbatecola AM, Barbieri M, et al. Evidence for anti-inflammatory effects of combined administration of vitamin E and C in older persons with impaired fasting glucose: impact on insulin action. J Am Coll Nutr. 2008. 27:505–511.

36. Sackesen C, Ercan H, Dizdar E, et al. A comprehensive evaluation of the enzymatic and nonenzymatic antioxidant systems in childhood asthma. J Allergy Clin Immunol. 2008. 122:78–85.

37. Schock BC, Young IS, Brown V, Fitch PS, Shields MD, Ennis M. Antioxidants and oxidative stress in BAL fluid of atopic asthmatic children. Pediatr Res. 2003. 53:375–381.

38. Scott DA, Poston RN, Wilson RF, Coward PY, Palmer RM. The influence of vitamin C on systemic markers of endothelial and inflammatory cell activation in smokers and non-smokers. Inflamm Res. 2005. 54:138–144.

39. Tagaya E, Tamaoki J. Mechanisms of airway remodeling in asthma. Allergol Int. 2007. 56:331–340.

40. Tecklenburg SL, Mickleborough TD, Fly AD, Bai Y, Stager JM. Ascorbic acid supplementation attenuates exercise-induced bronchoconstriction in patients with asthma. Respir Med. 2007. 101:1770–1778.

41. Tulic MK, Hamid Q. New insights into the pathophysiology of the small airways in asthma. Clin Chest Med. 2006. 27:41–52.

42. Wannamethee SG, Lowe GD, Rumley A, Bruckdorfer KR, Whincup PH. Associations of vitamin C status, fruit and vegetable intakes, and markers of inflammation and hemostasis. Am J Clin Nutr. 2006. 83:567–574.

43. Zhou J, Wolf CR, Henderson CJ, et al. Glutathione transferase P1: an endogenous inhibitor of allergic responses in a mouse model of asthma. Am J Respir Crit Care Med. 2008. 178:1202–1210.