THE major active ingredient of marijuana, Δ9-tetrahydrocannabi-nol (Δ9-THC), has been used as a psychoactive agent for thousands of years. Marijuana, and Δ9-THC, also exert a wide range of other effects including analgesia, anti-inflammation, immunosuppression, anticonvulsion, alleviation of intraocular pressure in glaucoma, and attenuation of vomiting1. The clinical application of cannabinoids has, however, been limited by their psychoactive effects, and this has led to interest in the biochemical bases of their action. Progress stemmed initially from the synthesis of potent derivatives of δ9-THC4,5, and more recently from the cloning of a gene encoding a G-protein-coupled receptor for cannabinoids6. This receptor is expressed in the brain but not in the periphery, except for a low level in testes. It has been proposed that the non-psychoactive effects of cannabinoids are either mediated centrally or through direct interaction with other, non-receptor proteins1,7,8. Here we report the cloning of a receptor for cannabinoids that is not expressed in the brain but rather in macrophages in the marginal zone of spleen.

Molecular characterization of a peripheral receptor for cannabinoids

The rapid recognition of intravenously injected colloidal carriers, such as liposomes and polymeric nanospheres from the blood by Kupffer cells, has initiated a surge of development for "Kupffer cell-evading" or long-circulating particles. Such carriers have applications in vascular drug delivery and release, site-specific targeting (passive as well as active targeting), as well as transfusion medicine. In this article we have critically reviewed and assessed the rational approaches in the design as well as the biological performance of such constructs. For engineering and design of long-circulating carriers, we have taken a lead from nature. Here, we have explored the surface mechanisms, which affords red blood cells long-circulatory lives and the ability of specific microorganisms to evade macrophage recognition. Our analysis is then centered where such strategies have been translated and fabricated to design a wide range of particulate carriers (e.g., nanospheres, liposomes, micelles, oil-in-water emulsions) with prolonged circulation and/or target specificity. With regard to the targeting issues, attention is particularly focused on the importance of physiological barriers and disease states.

Long-Circulating and Target-Specific Nanoparticles: Theory to Practice

A crucial question in the study of immunological reactions in the central nervous system (CNS) concerns the identity of the parenchymal cells that function as the antigen-presenting cells in that organ. Rat bone marrow chimeras and encephalitogenic, major histocompatability--restricted T-helper lymphocytes were used to show that a subset of endogenous CNS cells, commonly termed "perivascular microglial cells," is bone marrow-derived. In addition, these perivascular cells are fully competent to present antigen to lymphocytes in an appropriately restricted manner. These findings are important for bone marrow transplantation and for neuroimmunological diseases such as multiple sclerosis.

https://science.sciencemag.org/content/sci/239/4837/290.full.pdf

Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo.

Microglia-specific localisation of a novel calcium binding protein, Iba1

The present review summarizes recently acquired data in vivo, which support a role of CNS microglia as a source of defense cells in the CNS capable of carrying out certain immune functions autonomously. We have kept the following discussion restricted to microglial cells and have not included work on the immunological functions of astrocytes, which has been recently reviewed elsewhere (Fontana et al.: Immunological Reviews 137:3521-3527, 1987). Resting microglia are scattered uniformly throughout the CNS forming a network of potential immunoeffector cells, which can be activated by stimuli ranging from peripheral nerve injury over viral infections to direct mechanical brain trauma. The term "activated microglia" is used here to describe proliferating cells that demonstrate changes in their immunophenotype but have not undergone transformation into brain macrophages. Such a transformation can be stimulated by neuronal death but not by sublethal neuronal injury. Microglia may function as antigen-presenting cells and may thus represent the effector cell responsible for the recruitment of lymphocytes to the brain resulting in an inflammatory reaction. The recent developments in the understanding of microglial cell function may lead to a redefinition of the often cited "immune privilege" of the brain.

Functional plasticity of microglia: a review.

Two monoclonal antibodies, designated MRC OX-41 and MRC OX-42, have been shown to label subsets of macrophages. Using immunoperoxidase and immunofluorescence analysis, tissue macrophages were shown to be heterogeneous with respect to binding of MRC OX-41 and MRC OX-42 antibodies. Although both antibodies labelled subsets of macrophages, the antibodies also reacted with granulocytes and dendritic cells. The antigens recognized by these antibodies were identified by metabolic and cell surface labelling followed by immunoprecipitation and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). MRC OX-41 recognized a surface protein of 110,000-120,000 MW, while MRC OX-42 immunoprecipitated three polypeptides with molecular weights of 160,000, 103,000 and 95,000. The Fab fragment of MRC OX-42 antibody inhibited complement-mediated rosette formation between sensitized erythrocytes and rat macrophages and granulocytes. Membrane molecules with similar biochemical and functional properties to MRC OX-42 antigen have been identified in mouse and man as the receptors for iC3b, and it is probable that MRC OX-42 antibody recognizes the rat homologue of the receptors in these other species.

Macrophage heterogeneity in the rat as delineated by two monoclonal antibodies MRC OX-41 and MRC OX-42, the latter recognizing complement receptor type 3.

A mouse monoclonal antibody, MRC OX-43, has been shown to label vascular endothelium in all tissues of the rat except that of brain capillaries. Using immunoperoxidase staining, the antigen was shown to be expressed on the luminal surface of blood vessels. In addition, this antibody recognized a surface antigen on circulating erythrocytes and some macrophage populations, namely all those in the peritoneal cavity and a subset of alveolar macrophages. The antigen recognized by this antibody was identified on macrophages by metabolic and cell surface labelling followed by immunoprecipitation and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and found to be a surface protein of 90,000 MW.

MRC OX-43: a monoclonal antibody which reacts with all vascular endothelium in the rat except that of brain capillaries.

Introduction Human cytomegalovirus (HCMV) is a global health threat due to its ubiquity and lifelong persistence in infected people. During latency, host CD8+ T cell responses to HCMV continue to increase in a phenomenon known as memory inflation. We used murine CMV (MCMV) as a model for HCMV to characterize the memory inflation response to wild-type MCMV (KP) and a latency-defective mutant (ΔM33stop), which lacks M33, an MCMV chemokine receptor homolog. M33 is essential for normal reactivation from latency and this was leveraged to determine whether reactivation in vivo contributes to T cell memory inflation. Methods Mice were infected with wild-type or mutant MCMV and T cell responses were analyzed by flow cytometry at acute and latent time points. Ex vivo reactivation and cytotoxicity assays were carried out to further investigate immunity and virus replication. Quantitative reverse-transcriptase polymerase chain reaction (q-RTPCR) was used to examine gene expression during reactivation. MHC expression on infected cells was analyzed by flow cytometry. Finally, T cells were depleted from latently-infected B cell-deficient mice to examine the in vivo difference in reactivation between wild-type and ΔM33stop. Results We found that ΔM33stop triggers memory inflation specific for peptides derived from the immediate-early protein IE1 but not the early protein m164, in contrast to wild-type MCMV. During ex vivo reactivation, gene expression in DM33stop-infected lung tissues was delayed compared to wild-type virus. Normal gene expression was partially rescued by substitution of the HCMV US28 open reading frame in place of the M33 gene. In vivo depletion of T cells in immunoglobulin heavy chain-knockout mice resulted in reactivation of wild-type MCMV, but not ΔM33stop, confirming the role of M33 during reactivation from latency. Further, we found that M33 induces isotype-specific downregulation of MHC class I on the cell surface suggesting previously unappreciated roles in immune evasion. Discussion Our results indicate that M33 is more polyfunctional than previously appreciated. In addition to its role in reactivation, which had been previously described, we found that M33 alters viral gene expression, host T cell memory inflation, and MHC class I expression. US28 was able to partially complement most functions of M33, suggesting that its role in HCMV infection may be similarly pleotropic.

https://www.frontiersin.org/articles/10.3389/fimmu.2022.1047299/pdf

The CMV-encoded G protein-coupled receptors M33 and US28 play pleiotropic roles in immune evasion and alter host T cell responses

The salivary glands are a crucial site of replication for human cytomegalovirus (HCMV) and its murine counterpart, murine cytomegalovirus (MCMV). Studies of MCMV often involve the use of BALB/c strain mice, but most in vitro assays are carried out in the NIH 3T3 cell line, which is derived from Swiss Albino mice. This report describes a BALB/c-derived mouse salivary gland cell line immortalized using the SV40 large T antigen. Cells stained positive for PDGFR1 and negative for E-cadherin and PECAM-1, indicating mesenchymal origin. This cell line, which has been named murine salivary gland mesenchymal (mSGM), shows promise as a tool for ex vivo immunological assays due to its MHC haplotype match with the BALB/c mouse strain. In addition, plaque assays using mSGM rather than NIH 3T3 cells are significantly more sensitive for detecting low concentrations of MCMV particles. Finally, it is demonstrated that mSGM cells express all 3 BALB/c MHC class I isotypes and are susceptible to T cell-mediated ex vivo cytotoxicity assays, leading to many possible uses in immunological studies of MCMV.

/pdf/development-of-a-mouse-salivary-gland-derived-mesenchymal-2i8pg3du.pdf

Development of a mouse salivary gland-derived mesenchymal cell line for immunological studies of murine cytomegalovirus

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects the majority of the world population and causes lifelong latent infection. HCMV has been shown to exacerbate cardiovascular diseases, including myocarditis, vascular sclerosis, and transplant vasculopathy. Recently, we have shown that murine CMV (MCMV) recapitulates the cardiovascular dysfunction observed in patients with HCMV-induced myocarditis. To understand the viral mechanisms involved in CMV-induced heart dysfunction, we further characterized cardiac function in response to MCMV and examined virally encoded G-protein-coupled receptor homologs (vGPCRs) US28 and M33 as potential factors that promote infection in the heart. We hypothesized that the CMV-encoded vGPCRs could exacerbate cardiovascular damage and dysfunction. Three viruses were used to evaluate the role of vGPCRs in cardiac dysfunction: wild-type MCMV, a M33-deficient virus (∆M33), and a virus with the M33 open reading frame (ORF) replaced with US28, an HCMV vGPCR (i.e., US28+). Our in vivo studies revealed that M33 plays a role in promoting cardiac dysfunction by increasing viral load and heart rate during acute infection. During latency, ΔM33-infected mice demonstrated reduced calcification, altered cellular gene expression, and less cardiac hypertrophy compared with wild-type MCMV-infected mice. Ex vivo viral reactivation from hearts was less efficient in ΔM33-infected animals. HCMV protein US28 expression restored the ability of the M33-deficient virus to reactivate from the heart. US28+ MCMV infection caused damage to the heart comparable with wild-type MCMV infection, suggesting that the US28 protein is sufficient to complement the function of M33 in the heart. Altogether, these data suggest a role for vGPCRs in viral pathogenesis in the heart and thus suggest that vGPCRs promote long-term cardiac damage and dysfunction.

/pdf/the-viral-g-protein-coupled-receptor-homologs-m33-and-us28-35biy5gx.pdf

T M White

Papers

Macrophage heterogeneity in the rat as delineated by two monoclonal antibodies MRC OX-41 and MRC OX-42, the latter recognizing complement receptor type 3.

MRC OX-43: a monoclonal antibody which reacts with all vascular endothelium in the rat except that of brain capillaries.

The CMV-encoded G protein-coupled receptors M33 and US28 play pleiotropic roles in immune evasion and alter host T cell responses

Development of a mouse salivary gland-derived mesenchymal cell line for immunological studies of murine cytomegalovirus

The Viral G-Protein-Coupled Receptor Homologs M33 and US28 Promote Cardiac Dysfunction during Murine Cytomegalovirus Infection