Paul G. McMenamin

Cutting edge: Membrane nanotubes in vivo: a feature of MHC class II+ cells in the mouse cornea.

Membrane nanotubes are a recently discovered form of cellular protrusion between two or more cells whose functions include cell communication, environmental sampling, and protein transfer. Although clearly demonstrated in vitro, evidence of the existence of membrane nanotubes in mammalian tissues in vivo has until now been lacking. Confocal microscopy of whole-mount corneas from wild-type, enhanced GFP chimeric mice, and Cx3cr1gfp transgenic mice revealed long (>300 μm) and fine (<0.8 μm diameter) membrane nanotube-like structures on bone marrow-derived MHC class II+ cells in the corneal stroma, some of which formed distinct intercellular bridges between these putative dendritic cells. The frequency of these nanotubes was significantly increased in corneas subjected to trauma and LPS, which suggests that nanotubes have an important role in vivo in cell-cell communication between widely spaced dendritic cells during inflammation. Identification of these novel cellular processes in the mammalian cornea provides the first evidence of membrane nanotubes in vivo.

Body painting as a tool in clinical anatomy teaching.

The teaching of human anatomy has had to respond to significant changes in medical curricula, and it behooves anatomists to devise alternative strategies to effectively facilitate learning of the discipline by medical students in an integrated, applied, relevant, and contextual framework. In many medical schools, the lack of cadaver dissection as the primary method of learning is driving changes to more varied and novel learning and teaching methodologies. The present article describes the introduction and evaluation of a range of body painting exercises in a medical curriculum. Body painting was introduced into integrated clinical skills teaching sessions which included clinically important aspects of respiratory system, musculoskeletal system, and topics in regional anatomy including head and neck. Nontoxic body paints, easels, a mixture of brush sizes, and anatomical images were supplied. Students were allowed between 20 and 40 min to complete body painting tasks, in which they were encouraged to alternate between painting and acting as a model. Students were encouraged to use life‐like rendering and coloration where appropriate. Evaluation of these sessions was performed at the end of the semester as part of a larger evaluation process. The kinesthetic nature and active participation together with the powerful visual images of underlying anatomy appear to contribute to the value of body painting as a teaching exercise. In addition, it may have the added bonus of helping break down apprehension regarding peer–peer examination. Some practical advice on introducing this method of teaching in medical curricula based on the outcomes of the evaluation is given. On the basis of our experience and student feedback, we strongly advocate the use of body painting as an adjunct to surface anatomy and clinical skills teaching classes. Anat Sci Ed 2008.

Toll-like receptors at the ocular surface.

The Toll-like receptor (TLR) family of pathogen recognition molecules has an important role in recognizing microbial pathogens and microbial breakdown products. Activation of TLRs in the corneal epithelium induces CXC chemokine production and recruitment of neutrophils to the corneal stroma. Although essential for pathogen killing, neutrophils can cause extensive tissue damage, leading to visual impairment and blindness. In this review, we examine the role of TLRs in microbial keratitis and in noninfectious corneal inflammation, most commonly associated with contact lens wear. we present recent findings on TLR signaling pathways in the cornea, including MyD88- and TRIF-dependent responses and discuss the role of resident macrophages and dendritic cells. Finally, we examine the potential for targeting the TLR pathway as a potential therapeutic intervention for microbial keratitis and contact lens-associated corneal inflammation.

Differential turnover rates of monocyte-derived cells in varied ocular tissue microenvironments

Monocytes of bone marrow (BM) origin are circulating precursors that replenish dendritic cells and macrophage populations in peripheral tissues during homeostasis. The eye provides a unique range of varying tissue microenvironments in which to compare the different turnover rates of monocyte‐derived cells. This was investigated in the present study using radiation chimeras, whereby BM from Cx3cr1+/gfp mice was used to rescue myeloablated wild‐type (WT) BALB/c mice (conventional chimeras). The use of Cx3cr1+/gfp mice as BM donors allowed the clear visualization of newly recruited monocyte‐derived cells. Following BM reconstitution, mice were killed at 2, 4, 6, and 8 weeks, and wholemount ocular tissues were processed for immunohistochemistry and confocal microscopy. “Reverse” chimeras (WT into Cx3cr1+/gfp) were also created to act as a further method of cross‐referencing cell turnover rates. In conventional chimeras, Cx3cr1+/gfp cells began repopulating the uveal tract (iris, ciliary body, choroid) 2 weeks post‐transplantation with close to complete replenishment by 8 weeks. By contrast, the earliest recruitment of Cx3cr1+/gfp cells into the host retina occurred at 4 weeks. In reverse chimeras, a steady accumulation of host Cx3cr1+/gfp macrophages in the subretinal space of Cx3cr1+/gfp adult mice suggests that these cells arise from long‐term resident microglia and not newly recruited WT donor cells. In summary, chimeric mouse models, in which lineage‐specific cells carry a fluorescent reporter, have been used in the present study to visualize the turnover of monocyte‐derived cells in different tissue compartments of the eye. These data provide valuable insights into differential monocyte turnover rates within a single complex organ.

Stratification of Antigen-presenting Cells within the Normal Cornea

The composition and location of professional antigen presenting cells (APC) varies in different mucosal surfaces. The cornea, long considered an immune-privileged tissue devoid of APCs, is now known to host a heterogeneous network of bone marrow-derived cells. Here, we utilized transgenic mice that express enhanced green fluorescent protein (EGFP) from the CD 11c promoter (pCD11c) in conjunction with immunohistochemical staining to demonstrate an interesting stratification of APCs within non-inflamed murine corneas. pCD11c+ dendritic cells (DCs) reside in the basal epithelium, seemingly embedded in the basement membrane. Most DCs express MHC class II on at least some dendrites, which extend up to 50 μm in length and traverse up 20 μm tangentially towards the apical surface of the epithelium. The DC density diminishes from peripheral to central cornea. Beneath the DCs and adjacent to the stromal side of the basement membrane reside pCD11c–CD11b+ putative macrophages that express low levels of MHC class II. Finally, MHC class II–pCD11c–CD11b+ cells form a network throughout the remainder of the stroma. This highly reproducible stratification of bone marrow-derived cells is suggestive of a progression from an APC function at the exposed corneal surface to an innate immune barrier function deeper in the stroma.

Turnover of bone marrow-derived cells in the irradiated mouse cornea

In light of an increasing awareness of the presence of bone marrow (BM)‐derived macrophages in the normal cornea and their uncertain role in corneal diseases, it is important that the turnover rate of these resident immune cells be established. The baseline density and distribution of macrophages in the corneal stroma was investigated in Cx3cr1gfp transgenic mice in which all monocyte‐derived cells express enhanced green fluorescent protein (eGFP). To quantify turnover, BM‐derived cells from transgenic eGFP mice were transplanted into whole‐body irradiated wild‐type recipients. Additionally, wild‐type BM‐derived cells were injected into irradiated Cx3cr1+/gfp recipients, creating reverse chimeras. At 2, 4 and 8u2003weeks post‐reconstitution, the number of eGFP+ cells in each corneal whole mount was calculated using epifluorescence microscopy, immunofluorescence staining and confocal microscopy. The total density of myeloid‐derived cells in the normal Cx3cr1+/gfp cornea was 366u2003cells/mm2. In BM chimeras 2u2003weeks post‐reconstitution, 24% of the myeloid‐derived cells had been replenished and were predominantly located in the anterior stroma. By 8u2003weeks post‐reconstitution 75% of the myeloid‐derived cells had been replaced and these cells were distributed uniformly throughout the stroma. All donor eGFP+ cells expressed low to moderate levels of CD45 and CD11b, with approximately 25% coexpressing major histocompatibility complex class II, a phenotype characteristic of previous descriptions of corneal stromal macrophages. In conclusion, 75% of the myeloid‐derived cells in the mouse corneal stroma are replenished after 8u2003weeks. These data provide a strong basis for functional investigations of the role of resident stromal macrophages versus non‐haematopoietic cells using BM chimeric mice in models of corneal inflammation.

Bone Marrow Chimeras and c-fms Conditional Ablation (Mafia) Mice Reveal an Essential Role for Resident Myeloid Cells in Lipopolysaccharide/TLR4-Induced Corneal Inflammation

The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4−/− mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)+/C57BL/6 or eGFP+/TLR4−/− mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4−/− mice reconstituted with C57BL/6, but not TLR4−/− bone marrow cells donor cells were found to cause infiltration of eGFP+ cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP+ cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1α production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation.

Retinal Microglia and Uveal Tract Dendritic Cells and Macrophages Are Not CX3CR1 Dependent in Their Recruitment and Distribution in the Young Mouse Eye

PURPOSEnThe chemokine receptor CX3CR1 is expressed by monocyte-derived dendritic cells (DCs) and macrophages. CX3CR1 mediates leukocyte migration and adhesion in homeostatic and inflammatory conditions. Mice lacking Cx3cr1 have altered distribution and function of DC subpopulations in some tissue microenvironments. The present study compares the distribution of monocyte-derived cells in the normal retina and uveal tract as a prelude to the investigation of the role of CX3CR1 in murine models of ocular disease.nnnMETHODSnTransgenic mice in which either one (Cx3cr1 gfp/+, heterozygous) or both (Cx3cr1 gfp/gfp, homozygous) copies of the Cx3cr1 gene have been replaced by the enhanced green fluorescent protein (eGFP) reporter gene were used to investigate the role of Cx3cr1 expression on macrophages and DCs in the normal uveal tract and retina. Chimeric mice were used to investigate turnover of these cells in the normal, uninflamed eye.nnnRESULTSnConfocal analysis found no significant differences in the density, phenotype or morphology of eGFP+ cells between Cx3cr1 gfp/+ and Cx3cr1 gfp/+ mice in immunostained iris, ciliary body, or choroidal and retinal wholemounts. Flow cytometry also failed to detect any difference in the density or cell shape of eGFP+ cells between Cx3cr1 gfp/+ and Cx3cr1 gfp/+ mice. Chimeras revealed 73% turnover of monocyte-derived cells in the iris and 63% in the choroid by 6 weeks after transplantation.nnnCONCLUSIONSnThese data illustrate that homing or migration of DCs and macrophages to the uveal tract and retina in normal young mice is not Cx3cr1 dependent and provide a solid foundation for future studies of monocyte-derived cells and the role of Cx3cr1 in models of ocular disease.

CX3CL1/fractalkine regulates branching and migration of monocyte-derived cells in the mouse olfactory epithelium

The olfactory epithelium (OE) is a site of massive adult neurogenesis where olfactory sensory neurons (OSNs) are continuously turned over. Tissue macrophages have been implicated in phagocytosis of degenerating cells but the molecular mechanisms that allow for their recruitment while maintaining a neurogenic microenvironment are poorly understood. This study reports that the neuroprotective chemokine CX3CL1 is expressed by OSNs and olfactory ensheathing cells. Monocyte-derived cells in the OE depend on CX3CL1-signalling for intraepithelial migration and apical dendrite expression. These observations are first to demonstrate phenotypic differences in appearance and distribution of monocyte-derived cells in nervous tissue due to CX3CR1 deficiency.

The Monocyte Chemokine Receptor CX3CR1 Does Not Play a Significant Role in the Pathogenesis of Experimental Autoimmune Uveoretinitis

PURPOSEnTo examine the role of the monocyte chemokine receptor CX(3)CR1 in experimental autoimmune uveoretinitis (EAU).nnnMETHODSnEAU was induced in naive WT, Cx(3)cr1(gfp/+), and Cx(3)cr1(gfp/gfp) C57Bl/6 mice or chimeric mice. Ocular disease severity was graded by histologic analysis of resin sections. In addition, immunohistochemistry and confocal microscopy were performed on retinal whole mounts to characterize the monocytic infiltrate and changes in retinal microglia. To determine the relative roles of resident and blood-borne monocyte-derived cells in the active phase of uveoretinitis, EAU was induced 4 weeks after transplantation in chimeric mice (Cx(3)cr1(gfp/gfp)→WT and Cx(3)cr1(gfp/+)→WT), and analysis was performed at days 14, 16, 21, and 28 after immunization.nnnRESULTSnAfter EAU induction, disease scores were not significantly different in WT, Cx(3)cr1(gfp/+), and Cx(3)cr1(gfp/gfp) mice. Chimeric studies revealed both donor- and host-derived monocyte-derived cells in the inner retinal layers during early EAU; however, it was donor monocytic cells that infiltrated the photoreceptors, the site of the target antigen. The absence of CX(3)CR1 did not impede the ability of monocyte-derived cells from Cx(3)cr1(gfp/gfp) donor mice to infiltrate during the peak of EAU.nnnCONCLUSIONSnThe lack of CX(3)CR1 on monocyte-derived cells does not significantly influence the onset or severity of EAU. In addition, chimeric studies revealed that it is primarily blood-derived monocytes that mediate photoreceptor damage in the effector phase of EAU, and this process is not CX(3)CR1 dependent.