A Conduit System Distributes Chemokines and Small Blood-borne Molecules through the Splenic White Pulp

Access to the splenic white pulp is restricted to lymphocytes and dendritic cells. Here we show that movement of molecules from the blood into these confined areas is also limited. Large molecules, such as bovine serum albumin (68 kD), immunoglobulin G (150 kD), and 500 kD dextran are unable to enter the white pulp, whereas smaller blood-borne molecules can directly permeate this compartment. The distribution is restricted to a stromal network that we refer to as the splenic conduit system. The small lumen of the conduit contains collagen fibers and is surrounded in the T cell areas by reticular fibroblasts that express ER-TR7. It also contains the chemokine CCL21. Conversely, in B cell follicles the B cell–attracting chemokine CXCL13 was found to be associated with the conduit and absence of ER-TR7+ fibroblasts. These results show heterogeneity of reticular fibroblasts that enfold the conduit system and suggest that locally produced chemokines are transported through and presented on this reticular network. Therefore, the conduit plays a role in distribution of both blood-borne and locally produced molecules and provides a framework for directing lymphocyte migration and organization of the splenic white pulp

Porcine Sialoadhesin (CD169/Siglec-1) Is an Endocytic Receptor that Allows Targeted Delivery of Toxins and Antigens to Macrophages

Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that sialoadhesin is an endocytic receptor. In this report, we show that porcine sialoadhesin-specific antibodies and F(ab')2 fragments trigger sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the sialoadhesin endocytosis mechanism, two sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine sialoadhesin-specific immunotoxins efficiently kill sialoadhesin-expressing macrophages. Furthermore, porcine sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to sialoadhesin-expressing macrophages

Better preservation of the peritoneum in rats exposed to amino acid-based peritoneal dialysis fluid

BACKGROUND: Glucose-containing peritoneal dialysis fluids (PDF) show impaired biocompatibility, which is related partly to their high glucose content, presence of glucose degradation products, low pH, and lactate buffer, or a combination of these factors. In a rat chronic peritoneal exposure model, we compared effects of an amino acid-based PDF (AA-PDF) with a glucose-containing PDF on the peritoneal microcirculation and morphology. METHOD: Two groups of rats received 10 mL of either fluid daily for 5 weeks via peritoneal catheters connected to implanted subcutaneous mini vascular access ports. Leukocyte-endothelium interactions in the mesenteric venules were investigated by intravital microscopy. Quantification of angiogenesis and fibrosis and inspection of the mesothelial cell layer were performed by light and electron microscopy. RESULTS: Daily exposure to glucose-containing PDF resulted in a significant increase in the number of rolling leukocytes in mesenteric venules, whereas instillation of AA-PDF did not change the level of leukocyte rolling. Glucose-containing PDF evoked a significantly higher number of milky spots in the omentum, whereas this response was significantly reduced in animals exposed to the AA-PDF (p < 0.02). Chronic instillation of glucose-containing PDF induced angiogenesis in various peritoneal tissues, accompanied by fibrosis in the mesentery and parietal peritoneum. Quantitative morphometric evaluation of omentum and mesentery showed a clear trend toward less angiogenesis after treatment with the AA-PDF compared to the glucose-containing PDF, which reached statistical significance in the parietal peritoneum (p < 0.04). Instillation of AA-PDF resulted in approximately 50% reduction of fibrosis in the mesentery (p < 0.04) and approximately 25% reduction in the parietal peritoneum (p < 0.009) compared to glucose-containing PDF. Glucose-containing PDF damaged the mesothelial cell layer, whereas the mesotheium was intact after AA-PDF treatment, as evidenced by electron microscopy. CONCLUSION: Our data in a rat chronic peritoneal exposure model clearly demonstrate reduced immune activation (evidenced by decreased number of rolling leukocytes and decreased induction of omental milky spots) and reduced neoangiogenesis, fibrosis, and mesothelial damage of the peritoneal membrane after treatment with AA-PDF compared to glucose-containing PDF