Expression of different isoforms of TGF-beta and the latent TGF-beta binding protein (LTBP) by rat Kupffer cells

Kupffer cells (liver resident macrophages) make an important contribution to the perpetuation of liver diseases by synthesis and secretion of TGF-beta. In some cell types TGF-beta, is expressed as a large latent complex containing the latent TGF-beta binding protein (LTBP) in addition to the N-terminal TGF-beta precursor (latency associated peptide). This study aimed to identify LTBP expression in rat Kupffer cells.

Cells were isolated from rat liver by collagenase-pronase reperfusion, purified and cultured under standard conditions. TGF-beta and LTBP expression were characterized using alkaline phosphatase-anti-alkaline phosphatase immunostainings, reverse transcription-polymerase chain reaction and immunoprecipitation of metabolically labeled proteins.

Immunostainings of Kupffer cells with anti-sera against LTBP-1 (ab 39) and LTBP-2 indicated the expression of both LTBP isoforms in addition to the expression of latency associated peptide and TGF-beta. Transcripts of three LTBP isoforms (LTBP-1,-2,-3) and TGF-beta isoforms (TGF-beta-1,-2,-3) were detectable by reverse transcription-polymerase chain reaction. The LTBP-1D splice variant missing a part of the proteinase sensitive hinge region which has recently been described in hepatic stellate cells is expressed in Kupffer cells, too. Metabolic labeling of Kupffer cells with [35S]-Met/Cys followed by immunoprecipitation of the conditioned media using antisera against LTBP-1 and LTBP-2 indicated the secretion of high molecular mass TGF-beta complexes containing LTBP proteins of 230 and 170 kDa (LTBP-1) or 230 kDa (LTBP-2).

The results show that Kupffer cells partly synthesize and release TGF-beta as large latent complexes. This requires the extracellular activation of TGF-beta as a prerequisite for receptor binding and cellular signaling.