LITAF is readily detectable in ileal and colonic tissues from patients with either CD or UC, is significantly elevated above controls, and is localized to macrophages, a major source of TNF-alpha. These data provide strong evidence of a role for LITAF in the pathophysiological regulation of the TNF-alpha gene and underscore the potential value of anti-LITAF strategies in the clinical management of these diseases.

The liver is the most common site for metastasis by colorectal cancer, and numerous studies have shown a relationship between serum carcinoembryonic antigen (CEA) levels and metastasis to this site. CEA activates hepatic macrophages or Kupffer cells via binding to the CEA receptor (CEA-R), which results in the production of cytokines and the up-regulation of endothelial adhesion molecules, both of which are implicated in hepatic metastasis. Since tissue macrophages implicated in the metastatic process can often be difficult to isolate, the aim of this study was to develop an in vitro model system to study the complex mechanisms of CEA-induced macrophage activation and metastasis. Undifferentiated, human monocytic THP-1 (U-THP) cells were differentiated (D-THP) to macrophages by exposure to 200 ng/ml phorbol myristate acetate (PMA) for 18 h. Immunohistochemistry showed two CEA-R isoforms present in both U- and D-THP cells. The receptors were localized primarily to the nucleus in U-THP cells, while a significant cell-surface presence was observed following PMA-differentiation. Incubation of D-THP-1 cells with CEA resulted in a significant increase in tumor necrosis factor-alpha (TNF-alpha) release over 24 h compared to untreated D-THP-1 or U-THP controls confirming the functionality of these cell surface receptors. U-THP cells were unresponsive to CEA. Attachment of HT-29 cells to human umbilical vein endothelial cells significantly increased at 1 h after incubation with both recombinant TNF-alpha and conditioned media from CEA stimulated D-THP cells by six and eightfold, respectively. This study establishes an in vitro system utilizing a human macrophage cell line expressing functional CEA-Rs to study activation and signaling mechanisms of CEA that facilitate tumor cell attachment to activated endothelial cells. Utilization of this in vitro system may lead to a more complete understanding of the expression and function of CEA-R and facilitate the design of anti-CEA-R therapeutic modalities that may significantly diminish the metastatic potential of CEA overexpressing colorectal tumors.

Dysregulation of TNF-α in lamina propria macrophages (LPM) is a feature of inflammatory bowel diseases (IBD). LPS-Induced-TNF-Alpha-Factor (LITAF) is a transcription factor that mediates TNF-α expression. To determine whether LITAF participates in the mediation of TNF-α expression in acutely inflamed colonic tissues, we first established the TNBS-induced colonic inflammation model in C57BL/6 mice. LPM were harvested from non-inflamed and inflamed colonic tissue and inflammatory parameters TNF-α and LITAF mRNA and protein levels were measured ex-vivo. LPM from TNBS-treated mice secreted significantly more TNF-α at basal state and in response to LPS than LPM from untreated mice (p<0.05). LITAF mRNA and protein levels were elevated in LPM from TNBS compared with untreated animals and LPS further increased LITAF protein levels in LPM from inflamed tissue (P<0.05). To further confirm the role of LITAF in acutely inflamed colonic tissues, TNBS-induced colonic inflammation was produced in LITAF macrophage specific knockout mice (LITAF mac -/- mice) and compared to wild type (WT) C57BL/6. Twenty four hours following TNBS administration, colonic tissue from LITAF mac -/- mice had less MPO activity and reduced colonic TNF-α mRNA then WT C57BL/6 mice (p<0.05). LPM harvested from LITAF mac -/- secreted significantly less TNF-α in response to LPS than wild type (WT) C57BL/6 (p<0.05). This study provides evidence that LITAF contributes to the regulation of TNF-α in LPM harvested following acute inflammation or LPS treatment paving the way for future work focusing on LITAF inhibitors in the treatment of TNF-α-mediated inflammatory conditions.

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Dysregulation of TNF‐α secretion by lamina propria macrophages (LPM) is a hallmark of the inflammatory bowel disease Crohn's Disease (CD). LITAF has been shown to regulate TNF expression in macrophages and macrophage‐specific LITAF mac −/− mice (KO) are resistant to LPS induced sepsis. LITAF protein is increased by 30% in LPM isolated from C57BL/6 mice (WT) after TNBS administration, a well established model of CD (p<0.05). The aim of this study was to compare the colonic inflammatory responses of WT mice to LITAF mac−/− mice after administration of TNBS.Methods: WT and KO mice were administered TNBS (3 mg in 50% EtOH) rectally. After 24 hrs, body weights (BW) were determined and levels of the inflammatory marker myeloperoxidase (MPO) and TNF‐α mRNA were measured in colon tissue.Results: TNBS administration reduces BW in all animals compared to untreated controls (p<0.05); however, KO mice lost 50% less BW than the WT mice (p<0.05). MPO activity is five fold lower in KO mice compared to WT (p<0.05). Tissue levels of TNF‐α mRNA is also reduced in the KO compared to WT animals (376 ± 156 vs. 843± 253 copies) after TNBS administration.Conclusions:LITAF mac −/− have an attenuated inflammatory response to colonic TNBS administration. These results highlight a potential value of anti‐LITAF strategies for therapeutic management of CD. Research Support: BMC departments of Surgery and Pharmacology and NIDCR R01 DE014079.