COUTINHO, L. L.

NAKATA, L. C.

OLIVEIRA, M. C. de S.

REGITANO, L. C. de A.

ZAROS, L. G.

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8th World Congress on Genetics Applied to Livestock Production, August 13-18, 2006, Belo Horizonte, MG, Brasil QUANTITATIVE ANALYSIS OF BOVINE CYTOKINE mRNA  LEVELS IN RESPONSE TO TICK INFESTATION   L. C. Nakata1, L. G. Zaros2, M. C. S. Oliveira3, L. L. Coutinho2, L. C. A. Regitano3  1 UFSCar São Carlos Federal University, Rodovia Washington Luiz Km 235, São Carlos – S.P, 13.565-905, Brazil 2 ESALQ Agricultural Graduate School, University of São Paulo, Avenida Padua Dias 11, Piracicaba-S.P, 13.418-900, Brazil 3 EMBRAPA Southeast Cattle Research Center, Rodovia Washington Luiz KM 234, São Carlos-S.P, 13.560-970, Brazil   INTRODUCTION   Ticks cause economic losses to animal production in the order of billions of dollars annually (Horn, 1983). Alternatives for the control of these disease vectors are urgently needed due to the lack of efficacy and safety of the current methods that rely on chemicals (Pruett, 1999). Infestations with this parasite affect host immune responses, including several cytokines produced by T cells. The T cells, can be divided in TH1 and TH2. This classification is defined by the set of cytokines they secrete: TH1 cells secrete interleukin (IL)-1, IL-2, IL-8, IL-12, interferon-gama (IFN-γ) and monocyte chemoatractant protein 1 (MCP-1), while TH2 displays a cytokine secretion profile, including IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13 (Cher and Mosmann, 1987; Else and Finkelman, 1998). TH1 and TH2 lymphocytes subsets provide immunoregulatory signal for generation of cell-mediated and antibody responses, respectively (Wikel, 1996).  IL-8 and MCP-1 are chemokines and they act mainly like chemo-attractants for leukocytes, recruiting monocytes, neutrophils and other effector blood cells to infection sites. They are produced by monocytes, macrophages, fibroblasts and keratinocytes, and although both are produced by cells classified as TH1, MCP-1 has demonstrated to promote immunity TH2 (Janeway et al., 2002). IL-12 is secreted by macrophages as well as dendritic cells and is one of the cytokines produced early in several infections resulting in activation of natural killer cells (NK). IL-2 is a growth factor that guide proliferation and differentiation of T cells, and is produced for the own T cell activated. In mice infested with Rhipicephalus sanguineus tick, IL-2 and IFN-γ were found to be largely down-regulated, whereas IL-4 and IL-10 were up-regulated (Ferreira and Silva, 1999).  Many ticks modulate T-lymphocyte cytokine responses, resulting in a general pattern of down regulation of TH1 and an increase in TH2 cytokines (Schoeler and Wikel, 2001). Several methods exist that allow quantification of cytokine expression, such as real-time RT-PCR. In this sense, the present study used this methodology to quantify cytokines (IL-2, IL-4, IL-8, IL-12p35 and MCP-1-monocyte chemoatractant protein) in two groups of Nelore calves: one infestated (T) by ticks and other control (C), maintained free from infestation by ticks, in order to investigate their relationship with the immune response to Boophilus microplus infestations.   MATERIAL AND METHODS Animals and tissue samples. A group (T) of five Nelore calves were submitted to artificial infestation with Boophilus microplus ticks at five months of age, and a control group (C) was constituted of five Nelore calves maintained free from infestation during the experiment. Biopsies from skin and lymph node were collected from all animals at 9th day post infestation.  Real-time PCR for quantification of cytokine cDNA. Real-time PCR was performed using a Light Cycler, double-stranded DNA-binding dye Syber Green stain and the housekeeping 8th World Congress on Genetics Applied to Livestock Production, August 13-18, 2006, Belo Horizonte, MG, Brasil gene RPL-19 for normalization. The expression of interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 8 (IL-8), α35KDa subunit of interleukin 12 (IL-12p-35), and monocyte chemoatractant protein (MCP-1) were analyzed in skin and lymph node.  Statistical analyses. The comparative CT method was utilized for relative quantification. Data were analyzed by one-way analysis of variance followed by Student’s t-test. Differences between groups were considered significant if probability values of P< 0.1 were obtained.  RESULTS AND DISCUSSION The real-time PCR was used to assess cytokine profiles in cells extracted from skin and lymph nodes from Nelore calves. RPL-19, a housekeeping gene, was used for normalization because it did not have the expression altered by the treatment in both tissues studied (Table 1). In skin (Figure 1), MCP-1, IL-2, IL-8 and IL-12p35 quantification were not statistically different between the groups and no IL-4 transcription was detected in both groups. In lymph nodes (Figure 2), IL-2 and MCP-1 showed lower levels of mRNA (P<0.05 and P<0.1, respectively) in treated group, being IL-2 0.5-fold and MCP-1 0.3-fold less abundant in treated group in relation to control group. It is interesting to notice that IL-4, although not statistically significant (P<0.15), was 3-fold more abundant in treated group. No statistical differences were observed for IL-12p35 and IL-8 quantification from lymph nodes. Recent investigation has revealed that cytokines play a critical role in the prevention or progression of diseases. In this sense cytokine analysis is critical to research on disease resistance and on development of vaccine against several pathogens. Several previous reports showed that ticks modulate T-lymphocyte cytokine responses, resulting in a general pattern of down regulation of TH1 and an increase in TH2 cytokines (Ferreira and Silva, 1999; Schoeler et al., 2000; Mejri et al., 2001; Schoeler and Wikel, 2001). Kovář et al. (2002a,b) in a study about the impact of Ixodes ricinus tick salivary gland extract (SGE) on proliferation of mouse and human lymphocyte, found an impaired T-lymphocyte proliferation and a clear modulation of the immune response towards the TH2 pattern. They found that (SGE) increased the levels of IL-4 and reduced the levels of IL-2, a principal autocrine growth factor of TH1 lymphocytes, at both the protein and mRNA levels, similar to other data obtained in experiments with mouse cells (Ferreira and Silva 1999; Schoeler et al. 1999, 2000). In the early stages of infection, levels of IL-4 and IL-12 determine whether the cellular (TH1) or humoral (TH2) immune response will be activated (Wang et al., 1999). The results obtained in the present work mostly indicate an inhibition of the TH1 pattern, with the reduction of MCP-1 and IL-2 and a possible enhancement of TH2 through increase in IL-4 mRNA levels.  Table 1. Analysis of RPL-19 control gene. (S): Skin, (LN): Lymph node, (C): Control group, (T): Treated group. EP: Standard Error, CV: Variation coefficient.  Tissue Groups Mean±EP N CV(%) P-value C 19,33 ± 0,47 5 5,74 0,97 S T 19,35 ± 0,47 5 5,04        C 22,07 ±0,60 4 5,47 0,95 LN T 22,01 ± 0,53 5 5,25         8th World Congress on Genetics Applied to Livestock Production, August 13-18, 2006, Belo Horizonte, MG, Brasil -2,5-1,5-0,50,51,52,5MCP-1 IL-2 IL-8 IL-12       N-fold expression of treatment                  in relation to control group  Figure 1. Ratio of cytokine expression between treatment and control group in skin, normalized for a reference gene (RPL-19). Bars represent the means of cytokine mRNA abundance, expressed as the n-fold differences between groups (C and T) in relation to the calibrator (control group).   -1,5-0,50,51,52,53,5MCP-1 IL-2 IL-4 IL-8 IL-12++ *+      N-fold expression of treatment               in relation to control group  Figure 2. Ratio of cytokine expression between treatment and control (T/C) group in lymph node, normalized for a reference gene (RPL-19). Bars represent the means of cytokine mRNA abundance, expressed as the n-fold differences between groups (C and T) in relation to the calibrator (control group). *: P<0.05; ++: P<0.10; +: P<0.15  CONCLUSION The results showed that cytokines produced by TH1 response cells (IL-2 and MCP-1) had their expression down-regulated in lymph nodes from animals submitted to artificial infestation with Boophilus, while IL-4, a cytokine from TH2 response cells, although not statistically significant, was up-regulated in lymph nodes from treated animals, demonstrating a possible tendency for TH2 response. Additional studies are being accomplished to better understand the immune response of bovine host to ticks, the immune-regulatory cytokines and their potential use for diagnosis, prophylaxis and immune therapy.  REFERENCES Cher DJ , Mosmann TR. (1987) J. Immunol. 138:3688. Else KJ, Finkelman FD. (1998) Int. J. Parasitol. 28:1145-1158. Ferreira, B.R., Silva, J.S. (1999) Immunology.96:434–439. Horn, S.C. (1983) Bol. Def. Sanit. 2ed, Ministério da Agricultura, 79 pp. 8th World Congress on Genetics Applied to Livestock Production, August 13-18, 2006, Belo Horizonte, MG, Brasil Janeway CA, Tavers P, Walport M, Sholmchik.  (2002). « Imunobiologia: O Sistema Imune na saúde e na doença». Artmed, Porto Alegre, Rio Grande do Sul, Brasil. Kovář, L., Kopecký, J., Říhová, B. (2002). J. Parasitol. 87:1342–1348 a. Kovář, L., Kopecký, J., Říhová, B. B (2002). Parasitol. Res. 88:1066–1072 b. Mejri, N., Franscini, N., Rutti, B., Brossard, M. (2001). Parasite Immunol. 23:61–69. Pruett, J.H. (1999) Int. J. Parasitol. 29: 25-32. Schoeler, G.B, Manweiler, S.A, Wikel, S.K. (1999) Exp. Parasitol. 92:239–248. Schoeler, G.B, Manweiler, S.A., Wikel, S.K. (2000). Parasite Immunol. 22:31–40. Schoeler, G.B., Wikel, S.K.(2001) Ann. Trop. Med. Parasitol. 95: 755-771. Wikel SK (1996). Annu. Rev. Entomol. 41:1-22. 

Quantitative analysis of bovine cytokine mRNA levels in response to tick infestation.

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Quantitative analysis of bovine cytokine mRNA levels in response to tick infestation.

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