Parth B. Amin

Dose-Dependent Effects of Ethanol and E. Coli on Gut Permeability and Cytokine Production

INTRODUCTION The gut may prime inflammatory responses following shock/trauma insults. Ethanol (EtOH) use is common in trauma patients and may impair intestinal barrier function. We compared varying concentrations of EtOH on proinflammatory cytokine production of Caco2 cell monolayers and the resultant changes in barrier function. We hypothesized that even low concentrations of EtOH would cause significant cytokine release and barrier dysfunction in vitro. MATERIALS AND METHODS Confluent Caco2 cell monolayers were grown in a two-chamber culture system and exposed to varying concentrations of EtOH (0.1%, 0.5%, 1.0%, 1.5%, and 2.0%) with/without Escherichia coli C-25 (EC). Supernatants were collected and TNF and IL6 quantified by ELISA (pg/mL). Monolayer integrity was assessed by apoptosis and permeability measurements. RESULTS Caco2 production of TNF-alpha increased in a dose-dependent manner when incubated with increasing concentrations of EtoH. A synergistic effect was seen when E. coli was added to the apical chamber. A similar result was seen with the production of IL-6. A dose-dependent effect was also noted with EtOH with or without E. coli on apoptosis and permeability measurements. CONCLUSION In addition to alterations in gut permeability, increasing concentrations of ethanol have a synergistic effect with E. coli on Caco2 production of proinflammatory cytokines TNF and IL-6. The creation of a proinflammatory cytokine milieu with an altered barrier integrity may be a mechanism by which ethanol may increase septic complications in the injured patient.

Benign Fibroepithelial Polyp Arising in the Bronchus: A Case Report and Review of the Literature

Benign endobronchial polyps are rare findings that present a diagnostic dilemma not only for the clinician but also for the pathologist. We describe a man with repeated visits to emergency departments for coughing spells who ultimately underwent bronchoscopy and biopsy. The biopsy specimen was initially diagnosed as a leiomyoma, but the final pathologic diagnosis of the gross specimen was a benign fibroepithelial polyp. We present the clinical history of this patient along with a brief review of the published literature regarding this rare clinical entity.

The synergistic effect of ethanol and shock insults on Caco2 cytokine production and apoptosis

Gut epithelial cells are important in orchestrating immunoinflammatory responses in the gut and may impact systemic immunocompetent cells after shock and trauma. Ethanol (EtOH) intoxication is an important etiological factor in trauma and may increase the likelihood of posttraumatic septic complications. Both EtOH and gut I/R impair intestinal barrier function. However, their combined effects on intestinal epithelial cell function and barrier integrity are unknown. Confluent Caco2 cell monolayers were grown in a two-chamber culture system and exposed to 0.1% EtOH and/or Escherichia coli C-25 under normoxic (21% O2) or hypoxia (5% O2) followed by reoxygenation (H/R). Apical and basal compartment supernatants were collected, and TNF and IL-6 were quantitated by enzyme-linked immunosorbent assay (picograms per milliliter). Caco2 cell integrity was indexed by apoptosis and monolayer permeability. TNF-α production by Caco2 cells are greatest when incubated with EtOH and then exposed to H/R group. The apical levels of TNF production are consistently higher than basal levels, although the trend toward increased cytokine production is similar in both compartments. IL-6 production by the Caco2 cell is also greatest when Caco2 cells incubated with EtOH undergoes H/R. Lastly, the findings in apoptosis mirror the data of the TNF production in the apical compartment. Ethanol and H/R have a synergistic effect on cytokine production and barrier dysfunction in this model. They may also contribute to increased infectious complications and posttraumatic organ failure.

Secretory immunoglobulin A abrogates inflammatory responses and improves mortality after pseudomonas pneumonia.

BACKGROUND Secretory immunoglobulin A (SIgA) is the principle antibody at respiratory and other mucosal sites. Its concentration in mucosal secretions is influenced by route of nutrition and insults common to the trauma patient. SIgA has anti-inflammatory effects, which may protect against exaggerated inflammatory responses after infection. Polyimmunoglobulin receptor (pIgR) knockout (KO) mice, which do not have SIgA in mucosal secretions but are otherwise immunologically intact, were used to study the importance of SIgA in respiratory secretions after bacterial pneumonia. METHODS Pseudomonas aeruginosa or phosphate-buffered saline was administered intratracheally to pIgR-KO or conventional mice. Mortality was noted at 72 hours. Surviving animals were killed and blood, lung, and bronchoalveolar lavage (BAL) fluid samples were obtained and analyzed for myeloperoxidase, cytokine, and IgG levels. RESULTS Pneumonia was associated with increases in blood and bronchoalveolar lavage cytokine levels and increased lung myeloperoxidase activity. These results were significantly greater in pIgR-KO mice and associated with a threefold increase in mortality. CONCLUSION The importance of SIgA in maintaining mucosal homeostasis is demonstrated in this study, and the study supports efforts to preserve mucosal immune function in the trauma patient.

Cleavage of SIgA by gram negative respiratory pathogens enhance neutrophil inflammatory potential.

OBJECTIVE Secretory immunoglobulin A (SIgA), the principle immune defense at respiratory and other mucosal sites in the body, is highly dependant on its molecular structure for effective antibody function. Previous studies have demonstrated that gram-negative but not gram-positive isolates from patients with nosocomial pneumonia have IgA protease activity that contributes to the development of infection. We postulate that SIgA cleavage by bacteria would also affect anti-inflammatory properties of IgA and studied this in vitro. METHODS Sterile filtrates obtained from Pseudomonas, Acinetobacter, and methicillin resistant Staphylococcus aureus (MRSA) held in culture with SIgA were used to challenge polymorphonuclear neutrophils (PMNs) obtained from healthy volunteers. In a second group of experiments, blood monocytes were incubated with lipopolysaccharide (LPS) or LPS + IgA, and the resulting culture supernatants was used to stimulate PMNs in vitro. RESULTS LPS-stimulated monocytes increased CD11b expression, O2-generation and elastase release by PMNs. Secretory IgA but not IgG abrogated this response. Cleavage of SIgA by the gram-negative respiratory isolates, Pseudomonas aeruginosa and Acinetobacter baumanii also led to the loss of cellular effector function noted with intact SIgA. Additionally, PMN cytotoxic potential was similar to that noted with PMNs treated with supernatant from LPS-stimulated monocytes. CONCLUSION IgA cleavage by gram-negative respiratory isolates may lead to the development of pneumonia and the subsequent severity of the infection as a result of uncontrolled inflammatory responses by the host.

Secretory immunoglobulin a blunts gut-mediated priming of neutrophils in vitro.

BACKGROUND Gut ischemia may prime neutrophils to produce an exaggerated inflammatory response when challenged with bacterial pathogens. Secretory immunoglobulin A (sIgA) is the first line of defense against potential pathogens, but may also exert its anti-inflammatory effects on potentially destructive neutrophil functions. We hypothesized that sIgA would blunt the gut-mediated priming events that lead to neutrophil hypersensitivity to bacterial challenge. METHODS Confluent Caco2 cell monolayers were grown in a two-chamber culture system under normoxic or hypoxic conditions for 90 minutes followed by a 90-minute reoxygenation period. sIgA was placed in apical chamber media in experimental groups before reoxygenation period. Supernatants were then collected and incubated with neutrophils. Lipopolysaccharide was then used to activate neutrophils. Measurements of CD11b expression, elastase and superoxide anion production, and chemotaxis were undertaken. RESULTS Polymorphonuclear neutrophils (PMNs) treated with Caco2 cells undergoing hypox-reoxygenation followed by activation with lipopolysaccharide show a dramatic increase in inflammatory potential when compared with naïve neutrophils (n = 4, *p < 0.001). The addition of sIgA in this same group before the activation step showed a blunting of the inflammatory response, but never to the level of naïve PMN. CONCLUSIONS sIgA is the principal defense against potential pathogens at mucosal surfaces. Additional protective activity may be found in its ability to downregulate gut-mediated neutrophil priming. Although more work needs to be performed examining the role of sIgA in the pathogenesis of sepsis, this study shows that sIgA downregulates the measured determinants of neutrophil inflammatory potential.

The intestinal epithelial cell modulates the effect of alcohol on neutrophil inflammatory potential.

BACKGROUND Ethanol (EtOH) intoxication increases posttraumatic infectious complications. EtOH is thought to be immunosuppressive, but the effect of the interaction with the gut and associated microflora on host defense is unknown. We studied the ability of intestinal epithelial cells and bacteria to modulate EtOH effects on polymorphonuclear (PMN) cell function in vitro. METHODS Confluent Caco2 monolayers were exposed to 0.1% EtOH with or without Escherichia coli on the apical side of each chamber. Supernatants were collected from the basal side of the chambers and incubated with neutrophils. Controls were incubated with or without 0.1% EtOH alone. Chemotaxis, elastase, and superoxide anion release, and CD11b measurements were undertaken in these neutrophils and compared with controls. RESULTS Alcohol exhibited differing effects on the inflammatory potential of neutrophils in regards to CD11b expression, elastase production, and superoxide production. Direct effects of EtOH were anti-inflammatory, whereas indirect effects of EtOH on neutrophils modulated by Caco2 cells were proinflammatory. Chemotaxis was significantly reduced when neutrophils were directly treated with alcohol. CONCLUSION EtOH direct downregulates PMN inflammatory potential, but its impact of the intestinal epithelial cells produces a proinflammatory microenvironment for PMNs. The interaction of EtOH on the intestinal milieu may lead to neutrophil activation and increased organ injury after trauma in the intoxicated patient.

Sequence of immunoglobulin isotype exposure modulates inflammatory response to bacteria and lipopolysaccharide in vitro.

BACKGROUND Secretory immunoglobulin A (sIgA) and immunoglobulin G (IgG) are the principal immunoglobulins in the respiratory tract. Under normal circumstances, the upper respiratory tract contains predominantly sIgA, whereas IgG is of primary importance in the lower tract. Unlike other antibody isotypes, IgA antibodies participate in host defense functions without inciting inflammatory processes that might cause collateral damage to tissues. However, the ability of sIgA to modulate inflammatory reactions induced by other humoral factors is unclear. We examined the effect of the sequence of exposure to the two immunoglobulin isotypes on bacteria- or lipopolysaccharide (LPS)-mediated cytokine production by monocyte-polymorphonuclear neutrophil (PMN) cells in vitro. METHODS Blood monocytes were co-cultured with Escherichia coli or LPS. Either sIgA or IgG was added to subsets of cultures, which were incubated for 1 h at 37 degrees C. Culture supernatant liquids were then co-cultured with naïve PMNs for 1 h at 37 degrees C. Either IgG was added during this co-culture step or sIgA was added if IgG was added first. Cytokines were quantitated by enzyme-linked immunosorbent assay. RESULTS Significant increases in interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and IL-8 were noted after E. coli or LPS co-culture with monocytes and subsequent PMN challenge. Marked decreases in these pro-inflammatory cytokines were seen after the addition of sIgA or sIgA-IgG but not after IgG alone. This effect was more apparent with the immunoglobulin sequence IgA followed by IgG. CONCLUSION The sequence of immunoglobulin isotype involvement in infectious processes is important in modulating the cytokine response to bacteria and LPS by inflammatory cells. Our in vitro results support the critical role of sIgA in the proximal airways in mitigating inflammatory responses by other humoral defenses in the distal airways. Loss of effective sIgA function may contribute to increased morbidity and deaths from nosocomial pneumonia in compromised patients.