Dose-dependent immune response in milk cells and mammary tissue after intramammary administration of lipopolysaccharide in dairy cows

Abstract

The immune response in milk cells and the status of mammary tight junctions (TJ) in response to intramammary (IM) infusion of different doses of <i>Escherichia col</i>i lipopolysaccharide (LPS) was investigated. <i>Experiment I</i>: Seven German Braunvieh cows were IM infused into one quarter with 1 μg (LPS-1) and 3 μg (LPS-3) of LPS, respectively, and the contralateral control quarter with saline (9 g/l; C). Milk samples were taken immediately before and 12, 24, 36, 48, 60, 84 and 108 h after infusion and analysed for somatic cell counts (SCC), lactose, sodium (Na) and chloride (Cl) ions, and electrical conductivity (EC). Milk cell mRNA expression of various inflammatory factors was quantified by real-time RT-PCR. Blood samples were taken immediately after milking for the analysis of leukocytes (WBC), polymorphonuclear neutrophils (PMN), Na and Cl. Milk SCC, lactose, Na, Cl and EC did not differ significantly between LPS-1 and C quarters after the challenge. In LPS-3 quarters SCC levels increased within the first 12 h, reached peak levels between 12 and 36 h (<i>P</i> ≤ 0.001) and decreased (<i>P</i> ≤ 0.05) thereafter to reach baseline at 108 hours. Lactose in LPS-3 quarters decreased (<i>P</i> ≤ 0.05) to a minimum at 24 h and increased slightly thereafter while EC, Na, and Cl increased transiently in response to LPS-3. WBC and PMN levels in both groups decreased numerically within 24 h after LPS administration. In LPS-1, WBC at 24, 48 and 108 h were significantly lower whereas in LPS-3 they were significantly higher than at time 0. TNFα-mRNA expression in both groups did not change in response to IM LPS-challenge. IL-1β-mRNA expression at 12, 24 and 36 h in LPS-1 quarters increased significantly as compared to time 0. In LPS-3 quarters the mRNA expression values of all tested ILs increased significantly as compared to time 0 within 12 h after LPS-challenge. IL-1β-mRNA expression decreased (<i>P</i> ≤ 0.05) at 48 and 84 h in LPS quarters. IL-8 mRNA was significantly decreased at 84 h after challenge in LPS-3 quarters. COX-2-mRNA expression in LPS-1 quarters decreased significantly as compared to time 0 at 48, 84 and 108 h, with a minimum at 84 h (<i>P</i> ≤ 0.05). In LPS-3 quarters COX-2-mRNA levels increased (<i>P</i> ≤ 0.05) within 48 h after the LPS-challenge. <i>Experiment II</i>: Six cows (5 German Braunvieh, 1 Brown Swiss) were injected in one quarter with 100 μg LPS and in the contralateral quarter with saline (9 g/l; C). Mammary biopsy samples of both quarters were taken immediately before and at 3, 6, 9 and 12 h after infusion and mRNA expression of TJ proteins occludin (OCLN) and zonula occludens (ZO-) 1, 2 and 3 were quantified by real-time RT-PCR. OCLN-mRNA expression did not change in response to the IM infusion while that of ZO-1, ZO-2 and ZO-3 decreased significantly within six hours. In conclusion, a dose of 1 μg LPS did not initiate a immune response in the mammary gland. Furthermore the dose of 100 μg of LPS enhanced TJ permeability by reducing TJ plaque proteins density.