Proteomic evaluation of inflammatory proteins in rat spleen interstitial fluid and lymph during LPS-induced systemic inflammation reveals increased levels of ADAMST1.

The spleen is a part of the immune system and is involved in the response to a systemic inflammation induced by blood borne pathogens that may induce sepsis. Knowledge about the protein composition of the spleen microenvironment in a control situation and during systemic inflammation may contribute to our understanding of the pathophysiology of sepsis. To our knowledge, the proteome of the fluid phase of the spleen microenvironment has not previously been investigated. In order to access the proximal fluid surrounding the splenic cells, we collected postnodal efferent spleen lymph from rats by cannulation, and spleen interstitial fluid (IF) by centrifugation. The origin of the isolated spleen IF was assessed by the extracellular tracer (51)Cr-EDTA and the plasma tracer (125)I-HSA. Spleen lymph, IF, and plasma samples were collected during lipopolysaccharide (LPS) induced systemic inflammation and analyzed using a cytokine multiplex assay and, for the first time, using label-free mass spectrometry based proteomics. The concentrations of TNF-α, IL-1β, IL-6, and IL-10 increased severalfold in all fluids after LPS exposure. In total, 281, 201, and 236 proteins were identified in lymph, IF, and plasma, respectively, and several of these were detected after LPS only. A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) was detected by proteomics (the pro- region) in lymph only after LPS. ADAMTS1 was assessed by ELISA (the metalloproteinase domain), and the concentration was significantly higher in IF and lymph than in plasma in a control situation, showing local production in the spleen. A dramatic increase in ADAMTS1 was detected in lymph, IF, and plasma after LPS exposure. In conclusion, the procedures we used to isolate IF and lymph from the spleen during LPS enabled detection of locally produced proteins. Furthermore, we have demonstrated that the inflammatory proteome is different in the spleen microenvironment when compared to that in plasma.