Alemtuzumab mediates the CD39+ T-regulatory cell response via CD23+ macrophages.

Alemtuzumab (ALM) effectively prevents relapses of multiple sclerosis (MS). It causes lymphocyte depletion with subsequent enhancement of the T-regulatory cell population. Direct administration of ALM to T cells causes cytolysis. However, the T cells may be indirectly affected by monocyte-derived cells, which are resistant to ALM cytotoxicity. We aimed to examine whether ALM modulates monocytes and whether the crosstalk between monocytes and lymphocytes previously exposed to ALM would result in anti-inflammatory effects. The CD14+ monocytes of 10 healthy controls and 10 MS (treatment naive) patients were isolated from peripheral blood mononuclear cells (PBMCs), exposed to ALM and reintroduced to PBMCs depleted of CD14+ cells. The macrophage profile was assessed and T-cell markers were measured. ALM promoted M2 anti-inflammatory phenotype as noted by an increased percentage in the populations of CD23+ , CD83+ and CD163+ cells. The CD23+ cells were the most upregulated (7-fold, P = 0.0002), and the observed effect was higher in patients with MS than in healthy subjects. ALM-exposed macrophages increased the proportion of T-regulatory cells, without affecting the proportion of T-effector cells. Neutralizing the CD23+ monocytes with antibodies reversed the effect specifically on the CD4+ CD39+ T-regulatory cell subpopulation but not on the CD4+ CD25hi CD127lo FOXP3+ subpopulation. ALM induces the conversion of monocytes into anti-inflammatory macrophages, which in turn promotes T-regulatory cell enhancement, in a CD23-dependent manner. These findings suggest that the mechanism of action of ALM is relevant to aspects of MS pathogenesis.

TULP1 and TUB Are Required for Specific Localization of PRCD to Photoreceptor Outer Segments.

Photoreceptor disc component (PRCD) is a small protein which is exclusively localized to photoreceptor outer segments, and is involved in the formation of photoreceptor outer segment discs. Mutations in PRCD are associated with retinal degeneration in humans, mice, and dogs. The purpose of this work was to identify PRCD-binding proteins in the retina. PRCD protein-protein interactions were identified when implementing the Ras recruitment system (RRS), a cytoplasmic-based yeast two-hybrid system, on a bovine retina cDNA library. An interaction between PRCD and tubby-like protein 1 (TULP1) was identified. Co-immunoprecipitation in transfected mammalian cells confirmed that PRCD interacts with TULP1, as well as with its homolog, TUB. These interactions were mediated by TULP1 and TUB highly conserved C-terminal tubby domain. PRCD localization was altered in the retinas of TULP1- and TUB-deficient mice. These results show that TULP1 and TUB, which are involved in the vesicular trafficking of several photoreceptor proteins from the inner segment to the outer segment, are also required for PRCD exclusive localization to photoreceptor outer segment discs.

PRCD is concentrated at the base of photoreceptor outer segments and is involved in outer segment disc formation.

Mutations of the photoreceptor disc component (PRCD) gene are associated with rod-cone degeneration in both dogs and humans. Prcd is expressed in the mouse eye as early as embryonic day 14. In the adult mouse retina, PRCD is expressed in the outer segments of both rod and cone photoreceptors. Immunoelectron microscopy revealed that PRCD is located at the outer segment rim and that it is highly concentrated at the base of the outer segment. Prcd-knockout mice present with progressive retinal degeneration, starting at 20 weeks of age and onwards. This process is reflected by a significant and progressive reduction of both scotopic and photopic electroretinographic responses and by thinning of the retina, and specifically of the outer nuclear layer, indicating photoreceptor loss. Electron microscopy revealed severe damage to photoreceptor outer segments, which is associated with immigration of microglia cells to the Prcd-knockout retina and accumulation of vesicles in the inter-photoreceptor space. Phagocytosis of photoreceptor outer segment discs by the retinal pigmented epithelium is severely reduced. Our data show that Prcd-knockout mice serve as a good model for retinal degeneration caused by PRCD mutations in humans. Our findings in these mice support the involvement of PRCD in outer segment disc formation of both rod and cone photoreceptors. Furthermore, they suggest a feedback mechanism which coordinates the rate of photoreceptor outer segment disc formation, shedding and phagocytosis. This study has important implications for understanding the function of PRCD in the retina, as well as for future development of treatment modalities for PRCD deficiency in humans.

The progressive rod-cone degeneration (PRCD) protein is secreted through the conventional ER/Golgi-dependent pathway.

Retinitis pigmentosa (RP) is the most common form of hereditary retinal degeneration. Mutations of the PRCD gene are associated with RP in both dogs and humans. To date, four distinct PRCD mutations have been reported worldwide. Here we report the clinical phenotype of another patient with PRCD mutations, carrying the known p.R18X mutation and a novel missense mutation, p.P25T. This mutation affects a highly conserved amino acid, is predicted to be damaging by several prediction tools, and was not found in the public databases or in 115 ethnically-matched control individuals. The phenotype of this patient resembles that of previously reported patients with PRCD mutations, including bull's eye maculopathy, which appears to be a hallmark of the PRCD-induced phenotype. PRCD encodes for a 54 amino acids long protein with unknown function. The first 20 amino acids appear to encode for a signal peptide (SP), suggesting that PRCD is a secreted protein. To study PRCD secretion, C-terminally myc-tagged PRCD was expressed in cultured cells. Cells and conditioned media were analyzed by Western blot. PRCD was found in both cell extracts and media. However, a truncated PRCD protein lacking the first 20 amino acids was present only in cell extracts and not in media, confirming that PRCD extracellular secretion is mediated by its N-terminal SP. To characterize the secretory pathway of PRCD, various pharmacological agents which interfere with transport of proteins through the ER and Golgi to the plasma membrane were used. PRCD secretion was significantly inhibited by all tested pharmacological agents, confirming that it is secreted through the classic ER/Golgi-dependent secretory pathway. We tested the effect of two mutations on the PRCD protein, and found that p.C2Y, but not p.P25T, affects protein stability, and that neither mutation affects secretion. Our data suggest that PRCD functions as a secreted protein. These findings shed a new light on PRCD function and the etiology of RP.