Characterization of plasmacytoid dendritic cells in inflammatory arthritis synovial fluid.

OBJECTIVE: To examine the phenotype of dendritic cell subsets in synovial fluid and peripheral blood from patients with rheumatoid arthritis (RA) or spondyloarthropathy (SpA). METHODS: Multiparameter flow cytometry was used to identify and characterize dendritic cells in mononuclear cell populations isolated from synovial fluid and peripheral blood. RESULTS: Synovial fluid contained two subsets of dendritic cells (DC), myeloid and plasmacytoid. These subsets could also be identified in peripheral blood, but there were lower numbers of DC in peripheral blood compared with synovial fluid. Plasmacytoid DC were distinguished from the myeloid subset by high expression of CD123 and lack of expression of CD11c. In comparison with myeloid dendritic cells, the plasmacytoid subset were less mature, similar to those in peripheral blood. They failed to express CD83 and DC-LAMP, and had relatively low levels of CD40 and CD86. Comparison of dendritic cells in synovial fluid from RA and SpA patients showed increased numbers of the plasmacytoid subset in SpA. CONCLUSIONS: This is the first demonstration of the plasmacytoid subset of dendritic cells in synovial fluid. Since these cells are major producers of type I interferons, their increased numbers in SpA might be relevant to pathogenesis, but the immature phenotype in SpA synovial fluid may also indicate that conditions for maturation of this subset do not pertain in SpA synovium.

HLA-B27 expression does not modulate intracellular Chlamydia trachomatis infection of cell lines.

Chlamydia trachomatis is an obligate intracellular pathogen. Infection of susceptible individuals with this bacterium can trigger the development of reactive arthritis, an acute inflammation that is associated with the expression of the class I major histocompatibility antigen, HLA-B27. Other facultative intracellular pathogens, such as Yersinia and Salmonella spp., are also known triggers of reactive arthritis. Previous studies report conflicting results concerning whether the presence of HLA-B27 modulates the infection of cells with these enteric pathogens. In the present study, we have examined whether the expression of HLA-B27 can influence the infection of cell lines with C. trachomatis and also whether the replication of these bacteria is altered in HLA-B27-expressing cell lines. To do this, we have used a sensitive flow cytometric approach. We fixed and permeabilized cells and used fluorescein isothiocyanate-conjugated monoclonal antibody specific for chlamydia lipopolysaccharide to detect intracellular bacteria. The staining pattern obtained closely resembled the intracellular life cycle of chlamydia, with the appearance of brightly staining cells correlating to the microscopic detection of mature inclusion bodies. Moreover, since the percentage of cells that stained with the antibody was proportional to the infectious inoculum used, we were able to use the technique to quantitate the number of infectious organisms recoverable from infected cell lines. An important component of our study was the use of heparin to prevent reinfection of cells and thus enable the infection to be followed from a discrete time point. Our results suggest that HLA-B27 influences neither the infection nor replication of C. trachomatis serovar L2 within cell lines. Consequently, the role of HLA-B27 in the pathogenesis of reactive arthritis may lie downstream of the invasion and replication stages of the triggering pathogenic infection.

Differential effects of corticosteroids during different stages of dendritic cell maturation.

Dendritic cell (DC) maturation is a complex process involving many cell functions. We have studied how the exposure of DC to corticosteroids at different stages of DC maturation affects priming and the expansion of different subsets of CD4(+) T cells. Growth factor- dependent DC lines and fresh bone marrow-derived DC were used. When exposed to inflammatory stimuli, immature DC previously treated with dexamethasone were unable to undergo full maturation and were unable to prime Th1 cells efficiently. There was specific and significant reduction in the number of IFN-gamma-producing effector cell (shown by intracellular cytokine staining) and also in the amount of IFN-gamma produced. Interestingly, the number of IL-4-producing T cells and the amount of IL-4 synthesis was not significantly altered. Furthermore, multiple restimulation of T cells with these DC gave rise to a subpopulation of T regulatory cells (Tr1) which were negative for IFN-gamma and IL-4 but were IL-10 positive. In contrast, when DC were activated with lipopolysaccharide prior to dexamethasone treatment, the suppressive effect of glucocorticoids was not significant. Thus, the stage of DC maturation influences the inhibitory effect of corticosteroids. By arresting DC maturation, corticosteroids strongly reduce cell-mediated Th1 responses and allow the selective expansion of Tr1 cells.

Microglia induce myelin basic protein-specific T cell anergy or T cell activation, according to their state of activation.

Microglial cells are non-professional antigen-presenting cells (APC) the function of which is still controversial. Here, we studied the function of microglia derived from H-2(u) mice. We show that these microglia express a low level of B7.2 and CD40 and, interestingly, lack surface expression of B7.1. Resting and IFN-gamma-activated microglia were unable to activate naive and primed myelin basic protein (MBP)-specific CD4(+) T cells in the presence of MBP and encephalomyelitic MBP Ac1-11 peptide. Furthermore, in the presence of Ac1-11 peptide, CD4(+) TCR-transgenic T cells became anergized. Microglia became professional APC only after a multistep activation process involving both stimulation through cytokines [granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-gamma] and cognate signaling (B7-CD28 and CD40-CD40 ligand interactions). As such they were able to present MBP to both unprimed and primed T cells. Co-culture of microglia with GM-CSF up-regulated co-stimulatory molecules, in particular B7.1. Additional activation with IFN-gamma induced MHC class II and CD40 up-regulation. CD40-CD40 ligand interaction significantly enhanced microglial ability to prime TCR-transgenic T cells and was essential for presentation of MBP to in vivo primed non-transgenic T cells. We propose that microglia may serve different functions under different inflammatory conditions, depending on the cytokine milieu and the type of cognate interaction they are involved in.

Dendritic cells as natural adjuvants.

Dendritic cells (DCs) are professional antigen presenting cells that hold the key to the induction of T-cell responses. Therefore, the use of DCs for immunotherapy to stimulate immune responses has recently raised a great deal of interest. Many clinical trials using DCs have been initiated to stimulate immune responses against tumors or infectious agents. Several issues need to be considered before DCs can be used successfully as natural adjuvants: DCs have to be generated in sufficient numbers; they should display morphological, phenotypical, and functional properties of DCs; and they should be able to present antigens. In the present review we focus on methods for the purification of DCs from human bone marrow and peripheral blood and for the optimization of in vitro cell culture systems. Methods to generate growth factor-dependent mouse DC lines are also described.

Retroviral gene transfer, rapid selection, and maintenance of the immature phenotype in mouse dendritic cells.

We used the retroviral vector PINCO [which expresses the green fluorescent protein (GFP) as a selectable marker], to infect growth factor-dependent immature D1 dendritic cells (DC). The efficiency of infection in different experiments was between 5 and 30%, but subsequent cell sorting led to a virtually homogeneous population of GFP-positive cells. Retroviral infection did not modify the immature DC phenotype, as shown by the low expression of major histocompatibility complex and co-stimulatory molecules. Furthermore, the GFP-positive D1 cells underwent full maturation after lipopolysaccharide treatment, as indicated by a high expression of cell-surface MHC and co-stimulatory molecules, and also by strong stimulatory activity in allogeneic mixed lymphocyte reaction. The high efficiency of this retroviral system, the rapidity of the technique, and the possibility to overcome in vitro selection make this method very attractive for the stable introduction of heterologous genes into proliferating immature mouse D1 cells. Furthermore, this approach is suitable for functional studies of new DC-specific genes involved in DC maturation and survival.

Inflammation in the CNS: balance between immunological privilege and immune responses.

Inflammatory components play an important part in many diseases of the central nervous system (CNS). Recent evidence suggests that this may also be true of diseases which were previously considered as purely neuro-degenerative. However, it is also clear that inflammatory responses in the CNS differ in many ways from responses in non-CNS tissues. Some of these differences have been demonstrated by the use of animal models. For example, when bacteria are injected into the brain parenchyma, they induce a typical acute inflammatory response. However, unlike in other tissues, bacteria which are not cleared from the brain parenchyma remain undetected by the immune system. Some bacteria, such as bacillus Calmette-Guérin, can persist in the brain parenchyma for months sequestered in microglia and perivascular macrophages. When an animal with an intraparenchymal bacteria deposit is later sensitised peripherally, an immune response is evoked at the site of the deposits. The lesions induced in the CNS parenchyma are T-cell mediated and show characteristics typical of a delayed-type hypersensitivity response. The lesions produce a breakdown of the blood-brain barrier and demyelination. These immune responses are similar to those described for multiple sclerosis lesions. The responses to bacteria are unique to the brain parenchyma. Pathogens injected into the ventricles induce inflammatory responses similar to those in other non-CNS tissues: there is an acute inflammatory response which develops spontaneously into an immune mediated response within the first week.

Ultrastructural studies of an immune-mediated inflammatory response in the CNS parenchyma directed against a non-CNS antigen.

We have shown previously that heat-killed bacillus Calmette-Guerin injected into the brain parenchyma becomes sequestered behind the blood brain barrier for months undetected by the immune system. However, independent peripheral sensitization of the immune system to bacillus Calmette-Guérin results in recognition of bacillus Calmette-Guérin in the brain and the induction of focal chronic lesions [Matyszak M. K. and Perry V. H. (1995) Neuroscience 64, 967 977]. We carried out ultrastructural studies of these lesions. Prior to subcutaneous challenge we used immunohistochemistry to detect bacillus Calmette-Guérin which was found in cells with the morphology of macrophages/microglia and in perivascular macrophages. Eight to 14 days after subcutaneous challenge there was a conspicuous leucocyte infiltration at the site of bacillus Calmette-Guérin deposits within the brain parenchyma. The majority of these cells were macrophages and lymphocytes, with some lymphocytes showing characteristic blast morphology. Dendritic cells in close contact with lymphocytes were prominent. Inflammatory cells were found in perivascular cuffs and within the brain parenchyma. The tissue was oedematous and some axons were undergoing Wallerian degeneration with associated myelin degeneration. Throughout the lesions, but more commonly at the edges, we detected macrophages containing myelin in their cytoplasm close to intact axons and axons with evidence of remyelinating sheaths, suggestive of primary demyelination. In older lesions, two to three months after the peripheral challenge, the oedema was less pronounced and there was little evidence of Wallerian degeneration. There were still many macrophages. lymphocytes and dendritic cells, although the number of these cells was lower than in earlier lesions. Late lesions also contained many plasma cells which were not present in early lesions. In these late lesions there were bundles of axons with no myelin or a few axons with thin myelin sheaths, suggestive of persistent or ongoing demyelination or remyelination. These observations show that, during a delayed-type hypersensitivity lesion in the CNS, the leucocyte populations change with time, and suggest that the mechanisms and type of tissue damage are different in the early and late stages of the lesion.

Axonal damage in acute multiple sclerosis lesions.

One of the histological hallmarks of early multiple sclerosis lesions is primary demyelination, with myelin destruction and relative sparing of axons. On the other hand, it is widely accepted that axonal loss occurs in, and is responsible for, the permanent disability characterizing the later chronic progressive stage of the disease. In this study, we have used an antibody against amyloid precursor protein, known to be a sensitive marker of axonal damage in a number of other contexts, in immunocytochemical experiments on paraffin embedded multiple sclerosis lesions of varying ages in order to see at which stage of the disease axonal damage, in addition to demyelination, occurs and may thus contribute to the development of disability in patients. The results show the expression of amyloid precursor protein in damaged axons within acute multiple sclerosis lesions, and in the active borders of less acute lesions. This observation may have implications for the design and timing of therapeutic intervention, one of the most important aims of which must be the reduction of permanent disability.

Delayed-type hypersensitivity lesions in the central nervous system are prevented by inhibitors of matrix metalloproteinases.

We have studied the effect of an inhibitor of matrix metalloproleinases, BB-1101, on a delayed-type hypersensitivity (DTH) response in the CNS. We used a recently described model in which heat-killed bacillus Calmette-Guérin (BCG) sequestered behind the blood-brain barrier (BBB) is targeted by a T-cell mediated response after subcutaneous injection of BCG (Matyszak and Perry, 1995). The DTH lesions are characterised by breakdown of the BBB, macrophage and lymphocyte infiltration and tissue damage including myelin loss. Treatment with BB-1101, which is not only a potent inhibitor of matrix metalloproteinases but also strongly inhibits TNF-alpha release, dramatically attenuated the CNS lesions. Breakdown of the BBB and the recruitment of T-cells into the site of the lesion were significantly reduced. There were many fewer inflammatory macrophages in DTH lesions than in comparable lesions from untreated animals. There was also significantly less myelin damage (assessed by staining with anti-MBP antibody). The DTH response in animals treated with dexamethasone was also reduced, but to a lesser degree. No significant effect was seen after administration of pentoxifylline, a phosphodiesterase inhibitor with effects including the inhibition of TNF-alpha production. Our results suggest that inhibitors of matrix metalloproteinases may be of considerable therapeutic benefit in neuroinflammatory diseases.

The potential role of dendritic cells in immune-mediated inflammatory diseases in the central nervous system.

Dendritic cells of the rat were studied immunohistochemically with MRC OX62 monoclonal antibody and using electron microscopy. In normal CNS, a small number of OX62+ cells was detected in the choroid plexus and meninges. These cells were absent from other CNS and peripheral nervous system sites studied. Dendritic cells were also studied in two models of immune-mediated inflammatory conditions in the CNS. These were: acute experimental allergic encephalomyelitis and aberrant delayed-type hypersensitivity lesions induced as a response to heat-killed bacillus Calmette-Guérin sequestrated behind the blood-brain barrier. In addition, a group of animals with a delayed-type hypersensitivity response was treated with dexamethasone to assess the effect of steroid treatment on T-cells and OX62+ cells in CNS lesions. Dendritic cells were present in many but not all lesions in acute experimental allergic encephalomyelitis and their numbers were small. In experimental allergic encephalomyelitis lesions, dendritic cells were found predominantly in perivascular cuffs, where they constituted approximately 2% of the total number of major histocompatibility complex class II+ cells. Some of these cells were also detected in the CNS parenchyma, close to the perivascular cuff. In contrast, dendritic cells were present in all delayed-type hypersensitivity lesions studied. Their number in delayed-type hypersensitivity lesions was significantly higher than in experimental allergic encephalomyelitis lesions. Numerous OX62+ cells were found, even in three-month-old lesions. Electron microscopy studies revealed that these cells were often in close contact with lymphocytes. There was no significant change in the density of OX62+ cells, IL2R+ cells and OX19+ T-cells in delayed-type hypersensitivity lesions after seven-day treatment with dexamethasone, although there was a considerable reduction in the number of CD45RA+ T-cells. The high numbers of dendritic cells found in the delayed-type hypersensitivity lesions may be important in contributing to the chronicity of the response. They may also initiate autoimmune responses to CNS antigens uncovered during bystander tissue damage which occurs as a consequence of aberrant delayed-type hypersensitivity responses.

A comparison of leucocyte responses to heat-killed bacillus Calmette-Guérin in different CNS compartments.

We have previously shown that heat-killed bacillus Calmette-Guérin (BCG) injected into the CNS parenchyma does not produce a typical delayed-type hypersensitivity (DTH) response [23]. In this paper we have compared the initial leucocyte response in the CNS parenchyma, ventricles and skin to gain insight into the mechanisms by which the DTH response in the CNS might be controlled. We have found that 10(5) organisms of heat-killed BCG injected into either the CNS parenchyma or the lateral ventricles produced a rapid neutrophil response at the site of the injection, which was comparable with that in the skin. The neutrophil response resolved within the first week. Unlike the neutrophil response, the mononuclear phagocyte response in the CNS parenchyma was much smaller than that seen in the ventricles and the skin and it resolved within 4 weeks. Furthermore, the myelomonocytic response in the CNS parenchyma failed to clear the BCG. The acute inflammatory response in the choroid plexus/ventricles and skin developed with a similar time-course into a typical DTH response. After the first week, lesions at these two sites were composed predominantly of T-cells and macrophages. DTH lesions were still detected at both sites after 6 weeks. The failure of the immune system to recognize foreign antigens sequestrated in the CNS parenchyma may have significant implications especially in studies of inflammatory responses in the CNS of unknown origin.

Inflammation in the nervous system.

Earlier studies on inflammation in the CNS have largely focused on conditions with an immune component. Recent evidence has emerged, however, that the innate, acute inflammatory response in the CNS parenchyma is quite unlike that in other tissues. The meninges and ventricular compartments show more typical responses, as does the parenchyma of the brain in immature animals. It is becoming apparent that the cells of the mononuclear phagocyte lineage dominate inflammatory responses in the CNS parenchyma.

Demyelination in the central nervous system following a delayed-type hypersensitivity response to bacillus Calmette-Guérin.

We describe here a model of delayed-type hypersensitivity response in the CNS directed against a non-CNS antigen. The results presented in this paper show that bacillus Calmette-Guérin sequestrated behind the blood-brain barrier provokes an immune-mediated assault leading to bystander myelin damage. The delayed-type hypersensitivity response was induced by the intracranial injection of heat-killed bacillus Calmette-Guérin followed by subcutaneous immunization two to six weeks later. A single intracranial injection of bacillus Calmette-Guérin resulted in a rapid myelomonocytic response which persisted for approximately two weeks. By four weeks the inflammatory cells were no longer detected. Serum proteins were also excluded from the CNS parenchyma at this time. However, immunohistochemical staining with anti-bacillus Calmette-Guérin antiserum revealed the presence of bacillus Calmette-Guérin debris at the site of the original intracranial injection, indicating that the inflammatory response failed to clear the mycobacterium fully. Following peripheral sensitization with bacillus Calmette-Guérin in complete Freund's adjuvant, a strong delayed-type hypersensitivity response was detected at the site of bacillus Calmette-Guérin deposits in the CNS. An extensive inflammatory lesion was spread over a large area of the dorsal hippocampus. The lesion was composed predominantly of mononuclear phagocytes and T cells. Staining with anti-myelin basic protein antiserum showed bystander myelin damage. Delayed-type hypersensitivity responses were studied over several months and were still detected in the CNS five months after peripheral immunization.

Altered antigen expression of microglia in the aged rodent CNS.

Microglia, the resident macrophages of the central nervous system, are characterised by a highly specialized morphology and unusual antigenic phenotype. Microglia appear to be downregulated by their microenvironment when compared to other tissue macrophages. We have studied the microglia in brains of healthy, aged rats with a panel of monoclonal antibodies. We have found that microglia in the brains of these aged rats express antigens that are downregulated or absent from microglia of juvenile rats. The stimuli which give rise to this upregulated phenotype are not known. Age related changes in the phenotype of microglia should be taken into account when considering the possible role of microglia in neuropathological conditions.

Stromal macrophages of the choroid plexus situated at an interface between the brain and peripheral immune system constitutively express major histocompatibility class II antigens.

Using immunocytochemistry we have shown that there is a population of macrophages within the stroma of the choroid plexus of rats and mice which expresses high levels of major histocompatibility complex Class II antigens. In whole mount preparations of the choroid plexus, the morphology and regular distribution of these cells is similar to the Langerhans cells of the skin. These cells reside at an important interface between the central nervous system and the peripheral immune system and their possible role in immune-mediated diseases of the central nervous system is discussed.