Rosemary Dawson

Differentiation to the CCR2+ Inflammatory Phenotype In Vivo Is a Constitutive, Time-Limited Property of Blood Monocytes and Is Independent of Local Inflammatory Mediators

It is proposed that CCR2+ monocytes are specifically recruited to inflammatory sites, whereas CCR2− monocytes are recruited to normal tissue to become resident macrophages. Whether these subsets represent separate lineages, how differential trafficking is regulated and whether monocytes undergo further differentiation is uncertain. Using a mouse model of autoimmune uveoretinitis we examined monocyte trafficking to the inflamed retina in vivo. We show that bone marrow-derived CD11b+ F4/80− monocytes require 24 to 48 h within the circulation and lymphoid system before acquiring the CCR2+ phenotype and trafficking to the inflamed retina is enabled. This phenotype, and the capacity to traffic were lost by 72 h. Monocyte CCR2 expression followed a similar time course in normal mice indicating that differentiation to an inflammatory phenotype is a constitutive, time-limited property, independent of local inflammatory mediators. Phenotypic analysis of adoptively transferred cells indicated that circulating inflammatory monocytes also differentiate into CD11c+ and B220+ dendritic cells and F4/80+ tissue macrophages in vivo. Our data supports the hypothesis of continuous extravasation and progressive differentiation over time of inflammatory monocytes in the circulation rather than replication within the actively inflamed tissue, and supports the concept of myeloid dendritic cell differentiation from trafficking monocytes under physiological conditions in vivo.

Critical but divergent roles for CD62L and CD44 in directing blood monocyte trafficking in vivo during inflammation

Using noninvasive in vivo imaging and experimental autoimmune uveoretinitis as a model, we show for the first time that the mechanisms controlling blood monocyte recirculation through peripheral and lymphoid tissues alter during inflammation. The recirculation of monocytes in mice with ocular inflammation but not controls was found to depend on the selectin CD62-ligand (CD62L) and on CD44. Not only was rolling efficiency ablated or markedly reduced in antibody-treated mice, but most of the labeled monocytes also disappeared from the circulation within seconds, anti-CD44–treated monocytes homing to the lymph nodes and anti–CD62L-treated monocytes homing to the spleen. Our data indicate that, although PSGL-1 has a partial role in the transmigration of monocytes into the inflamed retina, CD62L has a key role in regulating recruitment of monocytes to lymphoid tissue from the blood during inflammation and that CD44 is required to maintain CD62L+ inflammatory monocytes within the circulation during inflammation. This effect was systemic, because sequestered monocytes accumulated in mesenteric as well as draining cervical lymph nodes, and inflammation dependent, because depletion of circulating blood monocytes was much reduced or absent in normal mice and accumulations of adoptively transferred monocytes in the lymphoid tissues did not occur.

Enhanced Tolerance to Autoimmune Uveitis in CD200-Deficient Mice Correlates with a Pronounced Th2 Switch in Response to Antigen Challenge

A single exposure to inhaled Ag 10 days before immunization leads to long term, Ag-specific tolerance. Respiratory tract myeloid APCs are implicated, but how regulation is invoked, and how tolerance is sustained are unclear. This study examines the in vivo function of the myeloid regulatory molecule CD200 in the process of tolerance induction. Despite earlier onset of experimental autoimmune uveitis in sham-tolerized, CD200-deficient mice, disease incidence and subsequent severity were actually reduced compared with those in wild-type mice. Protection was more effective and long term, lasting at least 28 days. Halting disease progression and tolerance in CD200−/− mice correlated with a marked increase in Th2-associated cytokine production by Ag-challenged splenocytes. Reduced overall disease and enhanced tolerance in the CD200-deficient mice in this model system were unexpected and may be related to altered populations of MHC class IIlow APC in the respiratory tract compared with wild-type mice together with associated activation of STAT6 in draining lymph nodes of tolerized mice. These data indicate that in the absence of default inhibitory CD200 receptor signaling, alternative, powerful regulatory mechanisms are invoked. This may represent either permissive dominant Th2 activation or an altered hierarchy of negative signaling by other myeloid cell-expressed regulatory molecules.

Effect of short-term macrophage depletion in the development of posterior capsule opacification in rodents.

Aim: To evaluate the role of macrophages in the development of posterior capsule opacification (PCO). Methods: For this purpose, an extracapsular lens extraction was performed in 18 consecutive Sprague-Dawley rats. Animals were treated with liposomal clodronate (Cl2MDP-lip-treated group, n = 10) or phosphate-buffered saline (PBS) (control group, n = 8) 1 day preoperatively and on the first day postoperatively, and sacrificed 3 days postoperatively. Masked clinical, light microscopy and immunohistochemistry studies were conducted. The Fisher exact test and randomisation test were used to assess statistically differences between groups. Results: A statistically significant reduction in the number of macrophages (ED1+, ED7+, ED8+) was found in the Cl2MDP-lip-treated group compared with the PBS-lip-treated group (p = 0.048, p = 0.004, p = 0.027, respectively). There were no statistically significant differences with regards to the presence/absence of central opacification (p = 0.29) and capsular wrinkling (p = 0.21) as detected clinically between groups. Similarly, a qualitative evaluation of the degree of PCO with regards to lens epithelial cell (LEC) proliferation, capsular wrinkling and Soemmerring ring formation showed no statistically significance between groups (p = 0.27, p = 0.061, p = 1.0, respectively). However, a statistically significant reduction in the number of lens epithelial cells (LEC) counted in the centre of the posterior capsule was found in the Cl2MDP-lip-treated group (p = 0.009). Conclusion: Depletion of macrophages was accompanied by a reduction in LEC in the centre of the posterior capsule in rodents.

Induction or suppression of a B cell-specific response to self antigen in vivo is dependent upon dendritic cell activation via the TNF-alpha receptor at the time of antigen uptake.

In this study we show that the retinal autoantigen, S‐antigen, contains a functional TNF‐α homologous domain which stimulates maturation and differentiation of cultured dendritic cells (DC) or tissue DC via the p55 TNF‐α receptor. Tissue DC became more dendritiform in shape, and migrated into culture supernatant. S‐antigen also stimulated accumulation of cell surface MHC class II antigen with a corresponding loss of acidic intracellular vesicles, and induced IL‐1β and IL‐12 mRNA expression in cultured bone marrow‐derived DC. In addition, cultured splenic DC primed immune responses to S‐antigen in vivo in the absence of other, exogenous cytokine sources. DC pulsed with either retinal S‐antigen or another retinal autoantigen, interphotoreceptor retinoid binding protein (IRBP), were able to stimulate naive T cell proliferation in vitro, but only S‐antigen‐pulsed DC were able to induce an immune response in vivo and initiate antibody class switching. In contrast, IRBP‐pulsed DC had no detectable in vivo priming effect and IgG antibody levels remained suppressed even after immunization with IRBP in complete Freunds adjuvant. These results indicate that DC from the same precursor population can either induce or suppress a B cell‐specific response to self antigen in vivo, the outcome being dependent upon DC activation at the time of antigen uptake and presentation.

UVEITOGENIC EPITOPES OF RETINAL S-ANTIGEN ARE GENERATED IN VIVO VIA AN ALTERNATIVE ANTIGEN-PRESENTATION PATHWAY

We have found that different antigen‐processing pathways are involved in the induction of experimental autoimmune uveoretinitis (EAU) by the retinal autoantigens S‐antigen and interphotoreceptor retinoid‐binding protein (IRBP). Although in vitro T‐cell proliferative responses to IRBP were completely inhibited in the presence of irreversible cysteine protease inhibitors, no significant reduction of S‐antigen proliferative responses was found. Furthermore, acidic proteolysis of S‐antigen by the cysteine protease cathepsin B prior to immunization radically reduced pathogenicity (disease severity). In addition, in vitro processing of S‐antigen, but not IRBP, was also found to be resistant to the action of cycloheximide and lysosomotropic agents, inhibition of proliferation only occurring after extended exposure of antigen‐presenting cells to methyl amine or high concentrations of chloroquine. These data indicate that an alternative pathway of antigen processing exists for S‐antigen, which is independent of processing within the normal endo‐lysosomal pathway and that uveitogenic peptides of naturally processed S‐antigen bind to major histocompatibility complex class II antigens either at the cell surface or within very early endosomes where cathepsin B is inactive.