Department of Rheumatology, Guy's Hospital, GKT School of Medicine, King's College London, London, UK
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Abstract |
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Methods. Paired lesional skin (SK) and synovial membrane (SM) from four PsA patients, paired SK and SM from four RA patients, SM from eight RA and eight OA patients, and normal SK from four volunteers were studied using standard immunohistochemistry.
Results. In all PsA and RA skin samples CD1-positive cells were abundantly detected both in the dermis and in the epidermis. However, in the 24 SM examined CD1-positive cells were rarely found. In one patient only with RA, a few CD1a-positive cells were found in the SM. CD1b was scarcely expressed in the lining layer (LL) of five SM and in very few cells in the sublining layer (SL) of 11 SM. CD1c was rarely expressed in the LL of six SM and in very few cells in the SL of 13 SM.
Conclusion. The paucity of CD1 in the PsA and RA synovium suggests that different subsets of antigen-presenting cells are involved in the pathogenesis of dermatitis and synovitis, respectively.
KEY WORDS: CD1, APC, Psoriatic arthritis, Rheumatoid arthritis
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Introduction |
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Another important element in the generation of a specific immune response is the type and nature of the antigen-presenting cells (APC) in various tissues. In the current study, therefore, we analysed the CD1 phenotype of APC in the skin and joint of patients with PsA. The five CD1 genes are located on chromosome 1 and are organized in a similar manner to the major histocompatibility complex (MHC) class I genes, with three exons encoding distinct extracellular domains which associate with ß2-microglobulin. This has been taken as an indication that CD1 and MHC class I and II genes evolved from a common ancestor [911]. However, at the amino acid level there is only limited sequence homology. CD1 molecules differ significantly in their function in that, while class I and II molecules present protein antigens, they have the unique capability of presenting non-protein antigen [12, 13]. The nature of these antigens in the case of CD1a has not been completely characterized, whereas CD1b and CD1c have been shown to present Mycobacterium tuberculosis (TB) lipid antigen [1416]. CD1 expression can be induced in vitro by culturing CD34+ haematopoietic progenitors and peripheral blood monocytes in the presence of granulocyte-macrophage colony stimulating factor (GMCSF) [17, 18].
Langerhans' cells (LC), the professional APC of the skin, are characterized by the strong expression of CD1 as well as by the presence of Birbeck granules [19]. Although Birbeck granule-positive Langerhans'-like cells have been described in the synovium [20], no data are yet available on the expression of CD1 by local APC. Therefore, the main aim of the work presented here was to examine the expression of CD1 molecules by synovial APC. We first chose to investigate patients with PsA as this is a unique disease which allows the study of the skin and synovial compartment within the same individual. The next question was to establish whether the findings observed in PsA were related to the disease-specific process or were due to a preferential localization of various APC programmed for different organs, skin and the joint, respectively. To this end, we investigated the expression of CD1 in the synovial membrane (SM) and in delayed-type hypersensitivity (DTH) reactions, induced by injecting tuberculin purified protein derivative (PPD), into the skin of patients with rheumatoid arthritis (RA). In addition, we used this model to try to establish if the increase of CD1-positive cells was due to an increased migration of these cells from the circulation rather than the result of an increased expression of the same molecules by resident cells.
In this paper we demonstrate that: (1) CD1 molecules are abundantly expressed in skin but not in the joints of patients with PsA; (2) similar results were obtained in DTH skin reactions and SM of patients with RA, suggesting that these findings are not disease specific; (3) the number of CD1-positive cells is increased in PsA lesional skin and in RA DTH reactions in comparison with normal skin; (4) the kinetics of localization of CD1-positive cells to DTH skin reactions (48 h) is compatible with a preferential migration from the circulation rather than an increased expression of this molecule by resident cells; and finally (5) factors (GM-CSF) known to induce the expression of CD1 cannot be the sole explanation for the profound differences found in the skin and in the synovium.
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Patients and methods |
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Biopsy samples
Biopsy samples from the active edge of psoriatic plaques and paired SM were obtained from four PsA patients referred to the out-patient department of the Department of Rheumatology, Guy's Hospital. SM biopsy specimens and paired skin samples, obtained from sites of PPD-induced DTH reactions, were also obtained from four RA patients. Skin samples were obtained using a 4 mm punch biopsy needle. The procedure was performed under aseptic conditions and local anaesthesia with 2% lignocaine. Synovial samples were obtained from the knee joint using a blind needle biopsy technique. Only knees with synovitis were biopsied and, if both knees were affected, the most affected was selected. The procedure was performed under aseptic conditions. Overlying skin and joint capsules were infiltrated with 2% lignocaine, and a Parker Pearson synovial biopsy needle was introduced into the knee by a lateral approach. Multiple synovial biopsies were obtained from the patellofemoral compartment from different sites in order to obtain a representative sample collection. Further synovial samples from eight RA and eight OA patients were obtained at the time of knee joint replacement. Four normal skin samples were also used as normal controls. Patients' informed consent and approval of the Ethics Committee were secured prior to performing sample collection.
Tissue preparation and staining
Synovial needle and skin punch biopsy specimens were embedded in optimal temperature cutting compound (OCT; Miles Laboratories, Elkhart, IN, USA) and snap frozen in isopentane cooled in liquid nitrogen. Samples were stored at -70°C until sectioned for immunohistological staining. Five micron thick sections were cut with a cryostat (Leitz, Wetzlar, Germany) at -22°C. Sequential sections were mounted on poly-L-lysine-coated slides and dried overnight at room temperature. Sections were fixed in acetone for 10 min, wrapped in aluminium foil, and stored at -70°C until further use. Cellular antigens, in both skin and SM, were identified by indirect immunoperoxidase technique on sequential sections. The monoclonal antibodies 7C6, 7C4 and 10C3, directed against CD1a, CD1b and CD1c, respectively, were kindly obtained from Dr W. Knapp [17]. GM-CSF staining was performed employing a mouse monoclonal antibody (5-315-8), a kind gift from Dr Ruedi (Sandoz, Basel, Switzerland).
Immunoperoxidase staining
Immunoperoxidase staining was performed using standard techniques, as previously described [21]. Briefly, after incubating with normal rabbit serum [1:20 in phosphate-buffered saline (PBS)] for 15 min, the primary monoclonal antibodies were applied for 1 h. After washing, sections were incubated for 30 min with a secondary antibody, horseradish peroxidase-conjugated rabbit anti-mouse (Dako, London), diluted 1:100 in PBS. The sections were developed in a solution of diaminobenzidine tetrahydrochloride (0.7 mg/ml) (Sigma, London) and counterstained with haematoxylin. Finally, sections were dehydrated by transferring through alcohol and CNP30, mounted in DPX compound (BDH, Poole, UK) and analysed with a Leitz microscope.
Controls
The staining of negative control samples was performed on all specimens studied. A procedure identical to that previously described was followed, except that the primary monoclonal antibody was substituted with an irrelevant antibody of the same isotype. No staining was noted in these sections.
Quantification
The results in the SM lining layer and sublining layer were expressed as the mean number of positive cells per microscopic field (x200). The results in skin were expressed as the mean number ± standard deviation (S.D.) of positive mononuclear cells overlying in the epidermis or underlying in the dermis 200 epidermal basal cells as described in detail by Furue et al. [22]. GM-CSF expression was evaluated as intensity of staining. Neg indicates no staining; +, mild; + + , medium; +++, strong staining.
Statistical analysis
The results were expressed as mean ± S.D. Statistical analyses were performed using the MannWhitney U-test.
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Results |
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Expression of CD1 in DTH skin reactions and synovium of patients with RA
In order to determine if the findings observed in PsA were disease specific or were common to any inflammatory process affecting skin and joints, we studied four paired DTH skin reactions and SM of patients with RA. The results for all patients studied can be seen in Table 2, while a representative example is shown in Fig. 1C
. It can be seen that the results were very similar to those obtained in PsA. A higher number of CD1a-positive cells was observed in the psoriatic epidermis compared with the rheumatoid DTH skin reactions, but this did not reach statistical significance. Except for one patient with RA highly responsive to PPD, similar numbers of dermal CD1a-positive cells were present in the two disease groups. No difference was found in the two disease groups regarding CD1b and CD1c expression, with the exception of the previously reported highly PPD responsive patient who showed a more intense infiltrate.
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Expression of CD1 in normal skin VS psoriatic and rheumatoid DTH reactions
To establish whether the number of CD1-positive cells in the skin increases during local inflammation, PsA and RA DTH skin samples were compared with normal non-inflamed skin. The number of CD1a-positive cells in healthy skin was much lower than the number in inflamed skin, confirming data already published [22]. In normal skin, the proportion of CD1-positive cells was as follows: 11.7 ± 2.2 in the epidermis and 12.5 ± 3.1 in the dermis (see Tables 1 and 2
for corresponding PsA and RA results, respectively). The comparison of normal skin with DTH skin reaction at 48 h allowed us also to draw some conclusions on the kinetics of localization of CD1-positive cells to a freshly induced inflammatory process and on the likely mechanism by which this occurs (see discussion below).
Expression of GM-CSF in PsA and RA skin and synovium
To investigate whether the lack of CD1 expression in the synovial compartment was possibly due to the lack of specific growth factors necessary to maintain its expression, we analysed GM-CSF production in all PsA and RA skin and synovial samples examined in the study. A representative example of a patient with PsA can be seen in Fig. 3. As expected from our previous published work [23], GM-CSF was detected in considerable amounts in the SM of all patients. The results, using a semiquantitative analysis method, are shown in Table 5
. It can be appreciated that GM-CSF was found as abundantly in the SM as in the paired skin samples.
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Discussion |
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CD1 molecules have the distinctive property of presenting non-protein antigen to T cells. They are expressed by professional APC such as LC and dendritic cells (DC) in several tissues such as skin, liver, kidney, lung and lymphoid organs [24, 25]. Therefore, it was long overdue to establish the level of expression of this set of molecules in a tissue such as the synovium, where APC are abundantly found [26, 27]. PsA was the best disease model to start such an analysis as high numbers of CD1-positive cells are known to be present in the skin of patients with Ps alone [28, 29] and where skin and joint inflammation coexist within the same individual. In addition, it is believed that both skin and synovial APC precursors originate from the bone marrow. An elegant confirmation of the bone marrow origin of LC has been obtained in female patients who received male bone marrow grafts, by the presence of Y chromosome-positive LC in the skin [30, 31]. Type A synoviocytes, at least in experimental animals, also originate from the bone marrow, as demonstrated in experiments in which bone marrow from beige mice was transplanted into irradiated normal mice [32]. Giant granules, similar to those seen in beige mouse synovial cells, were observed subsequently in the synovial lining cells of marrow recipients, indicating an influx of bone marrow-derived cells into the synovial lining [32]. In the bone marrow, DC and LC originate from common stem cells defined as dendritic/Langerhans' cell colony forming units (DL-CFU) [33]. Yet, despite the common origin, CD1-positive cells seem to be programmed to go to the skin but not to the joint. Furthermore, these cells appear to recirculate preferentially to the skin as indicated by experiments in which LC isolated from mouse skin and injected back into the animals preferentially locate into the same organ [34]. In this process, the CLA, a proposed skin homing receptor, appears to play a critical role [35]. CLA is a cell surface glycosylated protein which contains a CD15 (Lewisx) carbohydrate backbone and binds strongly to E-selectin [35]. This has led to the proposal of CLA and E-selectin as the homing-receptor/addressin-ligand pair specific for the skin [3638]. However, since E-selectin is expressed by many inflamed tissues including synovium where CLA+ cells are virtually absent [8], it is clear that the expression of this molecule per se is not sufficient to allow CLA+ cell extravasation to other tissues but the skin.
In addition to adhesion molecule interactions, other factors which are believed to be instrumental in the regulation of selective cell migration are a series of chemoattractant cytokines (chemokines, CK). CK are small molecular weight molecules (68120 amino acids in size) which mediate their effects via interactions with seven membrane-spanning domain receptors (CK-R), which signal through heterotrimeric GTP-binding proteins [3941]. It has been recently proposed that CK regulate the migration of DC to inflamed tissues and to the draining lymph nodes in a very sophisticated manner [42]. Immature DC, as well as their immediate monocyte precursors, express various receptors for inflammatory CK such as CCR1, 2, 5 and 6 which facilitate their migration to inflamed tissues [4345]. Once recruited to these sites, DC capture antigen and undergo a maturation process under the influence of pro-inflammatory cytokines, bacterial and viral products [46]. This together with the high local concentration of CK, induces a down-modulation of the inflammatory CK-R and the acquisition of specific CK-R such as CCR4, CCR7 and CXCR4 which facilitate the migration of DC into the local lymphatics and draining lymph nodes in response to cognate CK produced by lymphoid tissues. Therefore, it possible that various tissues produce specific CK which, in combination with specific adhesion molecules, regulate the selective trafficking of different APC to defined sites.
The second observation which was made in this study, namely the preferential localization of CD1-positive cells to the skin but not to the joint in DTH skin and SM of patients with RA, indicates that this is not a disease-specific phenomenon for PsA but rather a universal property of these cells. In this context, another important aspect to consider is the role of the local micro-environment. Interestingly, strongly CD1-positive purified LC show a considerable decrease or even loss of this molecule in culture in vitro [47]. However, CD1a expression can be readily maintained by co-culturing human LC with crude epidermal cell suspensions [48]. The importance of the local micro-environment is not unique to LC as, for example, it is known that in vitro culturing of endothelial cells, isolated from various tissues, leads to de-differentiation with changes in their original characteristics such as the loss of tight junctions in brain microvascular endothelium [49].
The increased accumulation of CD1-positive cells in DTH skin lesions at 48 h compared with normal skin, the third observation made in this paper, suggests that this phenomenon is probably due to a recruitment of these cells from the circulation rather than a neo-expression of CD1 molecules by resident cells. Although CD1 molecules can be induced in vitro in both CD34+ haematopoietic progenitors and circulating monocytes in the presence of GM-CSF, its expression does not take place significantly before day 37 of culture [17, 18]. Accepting all the limitations of extrapolating in vitro data to in vivo phenomena, it would appear that the timing at which DTH skin lesions were analysed would be too short to allow de novo expression of CD1 molecules by skin resident cells. Therefore, as already mentioned, these experiments support the concept that, during an inflammatory process, cells already programmed to migrate to the skin are preferentially recruited.
The final point which was considered in this study was the relationship between the production of regulatory molecules and CD1 expression. As mentioned earlier, one of the strongest inducers of CD1 is GM-CSF. Largely corroborating the results of the literature, our findings confirmed a strong production of GM-CSF in synovial biopsies of patients with PsA and RA which was comparable to the production in paired skin samples. Therefore, it would seem unlikely that a lack of GM-CSF production is responsible for the lack of CD1 molecules in the synovium. Of course, the technique used in our study is not informative as to the bio-activity of the GM-CSF detected. In addition, it is also possible that, while inhibitors of GM-CSF may be present in the synovium that prevent the expression of CD1 molecules, this tissue may be incapable of producing other factors which may be co-responsible for the induction of CD1 molecules.
In conclusion, in this study we provide evidence of the lack of CD1-positive cells in the synovium in PsA and RA which does not follow the strong presence of these cells in paired inflamed skin samples. It is likely that the interplay of local micro-environmental factors and the programmed migratory capacity of the cells themselves play a major role in the preferential localization of CD1-positive cells to the skin but not to the joint. Functional studies to address these points are clearly necessary to establish the real impact of these phenomena in the pathogenesis of skin and joint inflammation in PsA.
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Acknowledgments |
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Notes |
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References |
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