Primary ciliary dyskinesia (PCD) results from defects in motile cilia function. Mice homozygous for the mutation big giant head (bgh) have several abnormalities commonly associated with PCD, including hydrocephalus, male infertility, and sinusitis. In the present study, we use a variety of histopathological and cell biological techniques to characterize the bgh phenotype, and we identify the bgh mutation using a positional cloning approach. Histopathological, immunofluorescence, and electron microscopic analyses demonstrate that the male infertility results from shortened flagella and disorganized axonemal and accessory structures in elongating spermatids and mature sperm. In addition, there is a reduced number of elongating spermatids during spermatogenesis and mature sperm in the epididymis. Histological analyses show that the hydrocephalus is characterized by severe dilatation of the lateral ventricles and that bgh sinuses have an accumulation of mucus infiltrated by neutrophils. In contrast to the sperm phenotype, electron microscopy demonstrates that mutant respiratory epithelial cilia are ultrastructurally normal, but video microscopic analysis shows that their beat frequency is lower than that of wild-type cilia. Through a positional cloning approach, we identified two sequence variants in the gene encoding sperm flagellar protein 2 (SPEF2), which has been postulated to play an important role in spermatogenesis and flagellar assembly. A causative nonsense mutation was validated by Western blot analysis, strongly suggesting that the bgh phenotype results from the loss of SPEF2 function. Taken together, the data in this study demonstrate that SPEF2 is required for cilia function and identify a new genetic cause of PCD in mice.
Clever-1/Stabilin-1 is a scavenger receptor present on lymphatic and sinusoidal endothelium as well as on a subset of type II macrophages. It is also induced on vasculature at sites of inflammation. However, its in vivo function has remained practically unknown and this work addresses those unknown aspects. We demonstrate using in vivo models that Clever-1/Stabilin-1 mediates migration of T and B lymphocytes to the draining lymph nodes in vivo and identify the adhesive epitope of the Clever-1/Stabilin-1 molecule responsible for the interaction between lymphocytes and lymphatic endothelium. Moreover, we demonstrate that Ab blocking of Clever-1/Stabilin-1 efficiently inhibits peritonitis in mice by decreasing the entrance of granulocytes by 50%, while migration of monocytes and lymphocytes into the inflamed peritoneum is prevented almost completely. Despite efficient anti-inflammatory activity the Ab therapy does not dramatically dampen immune responses against the bacterial and foreign protein Ag tested and bacterial clearance. These results indicate that anti-Clever-1/Stabilin-1 treatment can target two different arms of the vasculature -traffic via lymphatics and inflamed blood vessels. IntroductionImmune cell trafficking between blood and lymphoid organs is an essential element in the proper functioning of the immune system. The mechanisms mediating lymphocyte entrance from the blood into the lymphoid organs and leukocyte trafficking to sites of inflammation are well known [1][2][3]. In contrast, mechanisms involved in lymphocyte migration from the periphery via the afferent lymphatics into the draining lymph nodes and their exit from the lymph nodes are poorly known. Besides lymphocytes, many tumors also use lymphatics for dissemination to distant sites of the body [4]. Eur. J. Immunol. 2009. 39: 3477-3487 DOI 10.1002 Innate immunity 3477Clever-1/Stabilin-1, also known as Feel-1, is a multifunctional molecule possessing scavenging ability on a subset of type II macrophages [5,6]. It is also present both on afferent and efferent lymphatic endothelial cells and sinusoidal endothelial cells in the liver and spleen [7][8][9][10]. In man, it is brightly expressed on high endothelial venules (HEV), which are the specific vessels within organized lymphatic tissues mediating lymphocyte entrance into the tissues, and on sinusoidal macrophages. Moreover, at sites of inflammation its expression is induced in flat-walled vessels. On lymphatic vasculature it is able to bind lymphocytes and certain tumor cells and on inflamed blood vessel endothelium it can mediate adhesion of lymphocytes, granulocytes and monocytes [8,11]. In vitro analyses have indicated it to be important in the transmigration step during the leukocyte extravasation process [12].Clever-1/Stabilin-1 is involved in two intracellular trafficking pathways, namely in receptor mediated endocytosis and recycling as well as in shuttling between endosomal compartment and trans-Golgi network. These complex mechanisms require interactions with several molecules ...
Stabilin-1/common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) is a multidomain protein present in lymphatic and vascular endothelial cells and type 2 immunosuppressive macrophages. In adults, stabilin-1/CLEVER-1 is a scavenging receptor and an adhesion molecule, but much less is known about its role during development. Here, we studied the expression and functions of macrophage stabilin-1/CLEVER-1 in human placenta and during human ontogeny. Using newly generated mAbs, we found that stabilin-1/CLEVER-1 is expressed on virtually all macrophages in term placenta, both in the decidua and in the placental villi. Placental stabilin-1/CLEVER-1 was involved in the scavenging of Ac-LDL (acetylated low density lipoprotein) and in the uptake of fluorescently labeled model antigen OVA. siRNA-mediated suppression of stabilin-1/CLEVER-1 altered the cytokine profile produced by placental macrophages. Stabilin-1/CLEVER-1 on placental macrophages mediated their adhesion to placental vessels and supported their transmigration through vascular endothelium. Finally, we found that stabilin-1/CLEVER-1 is induced very early in fetal macrophages, high endothelial venules, and lymphatic vessels in multiple lymphatic organs. Together, these data suggest that macrophage stabilin-1/CLEVER-1 can potentially regulate leukocyte migration and scavenging during the development of the placenta and fetus.
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