In an inflammatory state induced by HDE inhalation, it has been observed by our group that the HDE-induced inflammatory insult is followed by rapid PKCα activity, which stimulates TNFα release that induces IL-6 and PKCε stimulation concluding with a subsequent increase of IL-8 activity [17
] (). The observed data presented in this manuscript parallels this previously published sequential signaling. In accord with previously published manuscripts [26
], HDE exposure in our experiments caused robust statistical increases in inflammatory cell recruitment, mediator release, protein kinase C activity, and lung pathology development. In the HDE-only exposed group we observed increases in levels of IL-6, IL-8, TNFα, PKCε and PKA, all of which promote the inflammatory response. However, when alcohol was fed to HDE-exposed mice via the Meadows & Cook model, much of the HDE-induced inflammatory response was muted, or in the case of some cytokines, completely abrogated. These data suggest that the consumption of alcohol interrupts the inflammatory process by inhibiting signaling such as KC, MIP-2 (IL-8) and IL-6, along with down regulating the activity of PKCε in the co-exposed HDE + alcohol group. Alcohol-mediated protein kinase inhibition has been observed and cited in prior in vitro
-based publications [37
]. Our current data show that PKA inhibition by alcohol is also present in the in vivo
model. Interestingly enough, HDE exposure statistically increased PKCα levels in the lung tissues and tracheal epithelial cells of mice, however unlike PKCε and PKA, co-administration of alcohol and HDE did not reduce PKCα levels. This observation is key because PKCα has been shown to stimulate the cascade of signaling that regulate IL-6 release, PKCε and subsequent IL-8 release [17
]. Our data shows that alcohol consumption inhibits this PKCα initiated inflammatory response, and the lack of a subsequent KC/MIP-2 (IL-8) protein increase, under pro-inflammatory conditions, led us to conclude that the signaling disruption may be occurring between the PKCα activation and IL-6 vitiation. In our proposed sequential model [17
] (), TNFα represents an intermediate in the pathway between PKCα and chemokine release.
Proposed mechanism by which alcohol exposure ablates HDE-induced inflammation.
TNFα is a cytokine that mediates inflammatory responses via the NF-kB pathway. Previously, our group have shown that TNFα protein stimulates PKCε but not PKCα [17
], and subsequently TNFα stimulates IL-6 and IL-8 (KC/MIP-2) [19
], which in turn induces neutrophil migration to the site of inflammation [39
]. Even though total levels of BALF cells were statistically indifferent, in our data, neutrophil levels in the BALF were statistically lower as a proportion of total inflammatory cell counts in the alcohol + HDE group as compared to the HDE group. This is important because neutrophils are the first of the inflammatory cells to migrate towards the site of inflammation or injury [40
], and these neutrophils are called to the site of inflammation by the potent neutrophil chemokine, IL-8 (KC/MIP) [41
]. With alcohol attenuating HDE-stimulated release of IL-8 mouse homologues (KC/MIP-2), a reduction of neutrophils in BALF is an expected response [41
Peribronchiolar inflammatory cell aggregates normally generated by the mouse lung’s response to HDE were not as prevalent in the alcohol + HDE-exposed mice. This lack of cellular aggregates is likely mediated by decreased neutrophil activity. Neutrophils themselves are potent proinflammatory mediators [43
], and the effect of the absence of neutrophils in an in vivo
model under the influence of highly inflammatory insult such as HDE was previously unknown.
In our study, we observed 20% (2/10) mouse mortality. Even though n
= 10 is a small sample size, the statistical reduction of body weights in the co-exposed group shows that there was a level of general malaise in the group, which was unexpected and led us to speculate that alcohol’s interference in the function of TNFα in a high inflammatory environment could be a reason for these deleterious results observed in the alcohol + HDE co-exposed group. Alcohol has been shown to inhibit neutrophil cytokines including TNFα release [44
], IL-8, and NF-kB activation [45
]. According to Taieb et al
. 2002, this inhibition is, in part, attributed to alcohol’s ability to inhibit the expression and function of TNF converting enzyme (TACE) [44
]. TACE is an enzyme that proteolytically cleaves membrane bound TNFα allowing for soluble TNFα to be active. Pulmonary epithelial cells cannot mount a proper TNFα-mediated response to an inflammatory mediator such as HDE without soluble TNFα [46
]. These data have shown an inhibition of said TNFα-mediated response by alcohol in the presence of a robust inflammatory initiator. As a result under pro-inflammatory pressures, the inhibition of TACE will lead to increased levels of membrane-bound TNFα which could potentially stimulate some TNFα-mediated responses [47
], not the least of which could be cachexia [48
]. We acknowledge that serum TNFα levels, liver TNFα levels and an autopic pathological evaluation would be useful in elucidating the unexpected morbidity and mortality. Studies are being conducted to further characterize observations detailed in this manuscript. However with the weight loss and mortality data we have currently, it is possible to postulate that the mice in the co-exposed group were entering various levels of cachexia. This may seem to be contradictory to the TNFα data presented, however it has been observed that elevated levels of TNFα resulting from chronic alcohol can be attenuated with acute larger doses of alcohol [49
]. Concurrently it is well documented that alcohol reflux within the lungs due to the exhalation and condensation of alcohol onto the muscosal layer of the upper airways exposes the lung epithelial layer to concentrations of alcohol beyond what the lungs would be exposed to systemically. However, unlike the liver, the lung does not have robust stores of enzymes primed to metabolize compounds to allow for rapid excretion. As a result, direct pulmonary epithelium exposure is prolonged, leading to possibly a more acute response to alcohol exposure locally in the lung even under systemic chronic alcohol pressures. This acute response in a chronic exposure environment may be what is mediating the attenuated TNFα observed in the lung tissue.
Based on the published data and our data presented here, it is reasonable to propose that alcohol interferes with the proper HDE-induced inflammatory response possibly through the TACE/TNFα pathway, leading to reduced IL-6, PKCε, KC/MIP-2 activity, improper infiltrate clearance, weight loss, and mortality. Studies are currently underway to investigate the effect of alcohol on TNFα/TACE function during HDE exposure. Ultimately, these data relay the importance of a properly controlled proper inflammatory response, for when hog dust-induced inflammation is attenuated by alcohol consumption, C57Bl/6 mice have morbidity and mortality of which the etiology is currently unknown.