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Semaeva E, Tenstad O, Bletsa A, Gjerde EB, Wiig H. Isolation of rat trachea interstitial fluid and demonstration of local cytokine production in lipopolysaccharide-induced systemic inflammation. J Appl Physiol 104: 809-820, 2008. First published January 10, 2008 doi:10.1152/japplphysiol.00846.2007.-Access to interstitial fluid from trachea is important for understanding tracheal microcirculation and pathophysiology. We tested whether a centrifugation method could be applied to isolate this fluid in rats by exposing excised trachea to G forces up to 609 g. The ratio between the concentration of the equilibrated extracellular tracer 51 Cr-labeled EDTA in fluid isolated at 239 g and plasma averaged 0.94 Ϯ 0.03 (n ϭ 14), suggesting that contamination from the intracellular fluid phase was negligible. The protein pattern of the isolated fluid resembled plasma closely and had a protein concentration 83% of that in plasma. The colloid osmotic pressure in the centrifugate in controls (n ϭ 5) was 18.8 Ϯ 0.6 mmHg with a corresponding pressure in plasma of 22 Ϯ 1.5 mmHg, whereas after overhydration (n ϭ 5) these pressures fell to 9.8 Ϯ 0.4 and 11.9 Ϯ 0.4 mmHg, respectively. We measured inflammatory cytokine concentration in serum, interstitial fluid, and bronchoalveolar lavage fluid in LPS-induced inflammation. In control animals, low levels of IL-1␤, IL-6, and TNF-␣ in serum, trachea interstitial fluid, and bronchoalveolar lavage fluid were detected. LPS resulted in a significantly higher concentration in IL-1␤ and IL-6 in interstitial fluid than in serum, showing a local production. To conclude, we have shown that interstitial fluid can be isolated from trachea by centrifugation and that trachea interstitial fluid has a high protein concentration and colloid osmotic pressure relative to plasma. Trachea interstitial fluid may also reflect lower airways and thus be of importance for understanding, e.g., inflammatory-induced airway obstruction.interstitium; capillary filtration; extracellular matrix SIGNIFICANT INFORMATION ON fluid exchange and transport between the blood and tissue compartments may be gained from analysis of interstitial fluid, i.e., the fluid portion of the extracellular compartment. As an example, will proteins dissolved in the interstitial fluid exert a colloid osmotic pressure (COP) that opposes the corresponding pressure in plasma in such a manner that changes in protein content may impair the normally well-functioning and autoregulated fluid balance (4).In the airways, knowledge of the factors contributing to fluid homeostasis such as interstitial fluid COP is of vital importance because an edema in this organ may result in increased airway resistance and dyspnea (37, 44). Accordingly, access to interstitial fluid from the tracheobronchial tree is of major importance and may have therapeutical implications. In this context, fluid from trachea may be considered as a surrogate for lower airways. To our knowledge, only one study has dealt directly with the question of trachea interstitial fluid isolati...

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.

Vaccines using dendritic cells (DCs) harboring leukemic antigens to stimulate T cells is a possible treatment of acute myeloid leukemia (AML). Limitations of breaking tolerance to leukemic cells and lack of specific activation of T cell-mediated cytotoxicity may explain the discouraging clinical results with this approach. To break self-tolerance against AML cells, we loaded DCs with AML antigens and a bifunctional small interference (si) RNA targeting interleukin (IL) 10 and simultaneously activating toll-like receptors (TLRs). In vitro, this active siRNA inhibited (P < .05) IL-10 production by silencing the IL-10 gene in DCs. The active siRNA stimulated production of tumor necrosis factor alpha, implying activation of TLRs. Vaccination in a nonimmunogenic rat model mimicking human AML with the loaded DCs induced a substantial and specific T-cell cytotoxicity. Leukemic rats treated with the active siRNA lived longer and had markedly less leukemic cell mass infiltrating their bone marrow compared with rats given inactive siRNA (P < .05). Furthermore, compared with inactive siRNA treatment, the active siRNA led to significantly less extramedullar leukemic dissemination, evidenced by reduced matrix metalloproteinase activity and smaller spleens. Our data demonstrate that this bifunctional siRNA may work as an immunomodulatory drug with antileukemic properties.