Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure

<p>Abstract</p> <p>Background</p> <p>Bronchioles are critical zones in cigarette smoke (CS)-induced lung inflammation. However, there have been few studies on the <it>in vivo </it>dynamics of cytokine gene expression in bronchiolar epithelial cells in response to CS.</p> <p>Methods</p> <p>We subjected C57BL/6J mice to CS (whole body exposure, 90 min/day) for various periods, and used laser capture microdissection to isolate bronchiolar epithelial cells for analysis of mRNA by quantitative reverse transcription-polymerase chain reaction.</p> <p>Results</p> <p>We detected enhanced expression of keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) by bronchial epithelial cells after 10 consecutive days of CS exposure. This was mirrored by increases in neutrophils and KC, MIP-2, TNF-α, and IL-1β proteins in the bronchoalveolar lavage (BAL) fluid. The initial inhalation of CS resulted in rapid and robust upregulation of KC and MIP-2 with concomitant DNA oxidation within 1 hr, followed by a return to control values within 3 hrs. In contrast, after CS exposure for 10 days, this initial surge was not observed. As the CS exposure was extended to 4, 12, 18 and 24 weeks, the bronchiolar KC and MIP-2 expression and their levels in BAL fluid were relatively dampened compared to those at 10 days. However, neutrophils in BAL fluid continuously increased up to 24 weeks, suggesting that neutrophil accumulation as a result of long-term CS exposure became independent of KC and MIP-2.</p> <p>Conclusion</p> <p>These findings indicate variable patterns of bronchiolar epithelial cytokine expression depending on the duration of CS exposure, and that complex mechanisms govern bronchiolar molecular dynamics <it>in vivo</it>.</p

Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure-1

<p><b>Copyright information:</b></p><p>Taken from "Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure"</p><p>http://respiratory-research.com/content/9/1/7</p><p>Respiratory Research 2008;9(1):7-7.</p><p>Published online 21 Jan 2008</p><p>PMCID:PMC2248575.</p><p></p>ng sections were stained for oxidative DNA stress using an anti-8-OHdG antibody at 1 hr or 24 hrs following the last CS exposure. Normal mouse IgG1 in place of the 8-OHdG antibody served as a negative control . Images are representative of five mice per condition

Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure-9

<p><b>Copyright information:</b></p><p>Taken from "Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure"</p><p>http://respiratory-research.com/content/9/1/7</p><p>Respiratory Research 2008;9(1):7-7.</p><p>Published online 21 Jan 2008</p><p>PMCID:PMC2248575.</p><p></p> the plasma was measured using a quantitative enzyme immunoassay kit. Data represent average concentration of three mice per condition ± SEM. BAL fluids were collected at 24 hr after the last CS exposure and assayed for the presence of albumin using the albumin B test-Wako kit. Data represent the average concentration of eight mice per condition ± SEM. Statistical significance: ** = p < 0.01; *** = p < 0.001

Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure-8

<p><b>Copyright information:</b></p><p>Taken from "Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure"</p><p>http://respiratory-research.com/content/9/1/7</p><p>Respiratory Research 2008;9(1):7-7.</p><p>Published online 21 Jan 2008</p><p>PMCID:PMC2248575.</p><p></p>ed bronchiolar epithelium were determined by real-time RT-PCR. Data represent the average expression relative to β2-MG ± SEM from at least six mice. The part of data in Fig. 5A and 5B are also used for comparison. Statistical significance: * = p < 0.05; ** = p < 0.01; *** = p < 0.001 vs. before CS exposure at day 0

Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure-5

<p><b>Copyright information:</b></p><p>Taken from "Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure"</p><p>http://respiratory-research.com/content/9/1/7</p><p>Respiratory Research 2008;9(1):7-7.</p><p>Published online 21 Jan 2008</p><p>PMCID:PMC2248575.</p><p></p>xposure the time point before CS represents 24 hrs after 9 days exposure. At various times up to 24 hrs following last CS exposure, the bronchiolar epithelial cells were harvested by LCM and the expression of KC , MIP-2 , IL-1β , and TNF-α were determined by real-time RT-PCR. Data represent the average expression relative to β2-MG ± SEM from at least six mice. Statistical significance: *** = p < 0.001 vs. before CS exposure at each time point

Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure-4

<p><b>Copyright information:</b></p><p>Taken from "Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure"</p><p>http://respiratory-research.com/content/9/1/7</p><p>Respiratory Research 2008;9(1):7-7.</p><p>Published online 21 Jan 2008</p><p>PMCID:PMC2248575.</p><p></p>d IL-1β in whole lung homogenates and LCM-retrieved bronchiolar epithelium were determined by real-time RT-PCR. Data represent the average expression relative to β2-MG ± SEM from at least six mice. Statistical significance: * = p < 0.05; ** = p < 0.01; *** = p < 0.001

Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure-0

<p><b>Copyright information:</b></p><p>Taken from "Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure"</p><p>http://respiratory-research.com/content/9/1/7</p><p>Respiratory Research 2008;9(1):7-7.</p><p>Published online 21 Jan 2008</p><p>PMCID:PMC2248575.</p><p></p> the plasma was measured using a quantitative enzyme immunoassay kit. Data represent average concentration of three mice per condition ± SEM. BAL fluids were collected at 24 hr after the last CS exposure and assayed for the presence of albumin using the albumin B test-Wako kit. Data represent the average concentration of eight mice per condition ± SEM. Statistical significance: ** = p < 0.01; *** = p < 0.001

Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure-6

<p><b>Copyright information:</b></p><p>Taken from "Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure"</p><p>http://respiratory-research.com/content/9/1/7</p><p>Respiratory Research 2008;9(1):7-7.</p><p>Published online 21 Jan 2008</p><p>PMCID:PMC2248575.</p><p></p>was identified as described in Materials and Methods. Data represent the average number of total cells and neutrophils per ml BAL fluid ± SEM from eight mice. The data set of Fig. 3A and 3C are also included for comparison. Statistical significance: * = p < 0.05; ** = p < 0.01; *** = p < 0.001 vs. before CS exposure at day 0

Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure-7

<p><b>Copyright information:</b></p><p>Taken from "Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure"</p><p>http://respiratory-research.com/content/9/1/7</p><p>Respiratory Research 2008;9(1):7-7.</p><p>Published online 21 Jan 2008</p><p>PMCID:PMC2248575.</p><p></p>AL fluid were determined by ELISA. Data represent the average concentration per ml BAL fluid ± SEM from eight mice. The data set of Fig. 4A and 4B are shown for comparison. Statistical significance: * = p < 0.05; ** = p < 0.01; *** = p < 0.001 vs. before CS exposure at day 0