Platelet CLEC-2 protects against lung injury via effects of its ligand podoplanin on inflammatory alveolar macrophages in the mouse

There is no therapeutic intervention proven to prevent acute respiratory distress syndrome (ARDS). Novel mechanistic insights into the pathophysiology of ARDS are therefore required. Platelets are implicated in regulating many of the pathogenic processes that occur during ARDS; however, the mechanisms remain elusive. The platelet receptor CLEC-2 has been shown to regulate vascular integrity at sites of acute inflammation. Therefore the purpose of this study was to establish the role of CLEC-2 and its ligand podoplanin in a mouse model of ARDS. Platelet-specific CLEC-2-deficient, as well as alveolar epithelial type I cell (AECI)-specific or hematopoietic-specific podoplanin deficient, mice were established using cre-loxP strategies. Combining these with intratracheal (IT) instillations of lipopolysaccharide (LPS), we demonstrate that arterial oxygen saturation decline in response to IT-LPS in platelet-specific CLEC-2-deficient mice is significantly augmented. An increase in bronchoalveolar lavage (BAL) neutrophils and protein was also observed 48 h post-IT-LPS, with significant increases in pro-inflammatory chemokines detected in BAL of platelet-specific CLEC-2-deficient animals. Deletion of podoplanin from hematopoietic cells but not AECIs also reduces lung function and increases pro-inflammatory chemokine expression following IT-LPS. Furthermore, we demonstrate that following IT-LPS, platelets are present in BAL in aggregates with neutrophils, which allows for CLEC-2 interaction with podoplanin expressed on BAL inflammatory alveolar macrophages. Taken together, these data suggest that the platelet CLEC-2-podoplanin signaling axis regulates the severity of lung inflammation in mice and is a possible novel target for therapeutic intervention in patients at risk of developing ARDS. </jats:p

Studies on function and regulation of the transcription factor C/EBPalpha

The transcription factor C/EBPalpha was originally identified as a protein involved in hepatocyte-specific gene regulation. Later, it has been demonstrated that C/EBPalpha is expressed in severalother tissues, mainly in adipose cells, lung, intestine, skin and in myeloid cells. C/EBPalpha takespart in cell-specific gene expression in these different cell types and has been suggested to bean important regulator of the terminal differentiation process, most clearly demonstrated inadipocytes. An antiproliferative effect of C/EBPalpha has also been observed in this cell type.This thesis was aimed at further elucidation of the function of the C/EBPalpha protein, by usingdifferent rodent in vivo systems. Furthermore, the aim was to increase the knowledge aboutthe transcriptional regulation of the C/EBPalpha gene itself.Our studies performed on the C/EBPalpha promoter by footprinting and gel-shift experiments,demonstrated 6 factor binding sites when an adult rat liver nuclear extract was used. Sites forUSF and C/EBP were identified. Transfection experiments showed that USF is the mostimportant transactivator within the -350/+3 promoter fragment and that both C/EBPalpha itselfand C/EBPbeta are able to transactivate this promoter. Expression studies of C/EBPalpha bothduring rat liver regeneration, after partial hepatectomy, and during chemically induced ratliver carcinogenesis, demonstrated a decreased expression of the C/EBPalpha gene attranscriptional, mRNA and protein levels . The observation that C/EBPalpha expression decreasesduring hepatocyte proliferation is in line with the previously suggested antimitotic functionof the protein. To obtain more information about the importance of C/EBPalpha in vivo weperformed an inactivation of the gene in mice by homologous recombination in ES cells. Micedeficient in the C/EBPalpha protein show normal embryonic development, but dramaticphenotypical effects appear soon after birth. Thus, although apparently normal at birth, C/EBPalpha knockout mice die 7-10 hours postpartum, probably from hypoglycemia, since bloodglucose levels are extremely low at the time of death. Histological analysis revealed very lowlevels of stored glycogen in the liver and low levels of fat in adipose tissues and liver innewborn knockout mice. These results suggest that C/EBPalpha is crucial for the regulation ofgenes involved in perinatal glycogen and fat synthesis in liver and adipose tissues. Histologicalanalysis of lungs from C/EBPalpha knockout mice, demonstrated a retarded pulmonarydevelopment and hyperproliferation of alveolar type II cells. Interestingly, about 20% of thenewborn knockout mice showed severe respiratory problems, often resulting in death justsome minutes after birth. The fact that type II cells are producers of surfactants, lipoproteinsof importance in the alveolar inflation process, and that there is a known temporal correlationbetween C/EBPalpha expression and surfactant production, makes it possible that the observedrespiratory dysfunction is due to inadequate surfactant levels in some of the C/EBPalpha knockoutmice. Northern analysis of liver RNA demonstrated a decrease in albumin and an increase ina-fetoprotein mRNA levels. The levels of b-actin, c-jun and c-myc mRNA were all increasedin livers from knockout mice. Furthermore, a 5-10 times increase in the frequency ofhepatocytes producing the S-phase associated PCNA protein was observed. Taken together,these results clearly indicate a less differentiated status and an increased proliferative activityin hepatocytes deficient in the C/EBPalpha protein.In conclusion, the results presented in this thesis, suggest that C/EBPalpha is an important factorin vivo for the acquisition of crucial differentiated functions in liver, adipose tissues andprobably also in the lung. Furthermore, an antiproliferative role in hepatocytes is suggested.ISBN 91-628-2039-