Macrophage subpopulations in rheumatoid synovium: reduced CD163 expression in CD4+ T lymphocyte-rich microenvironments.

OBJECTIVE: The cell surface glycoprotein CD163 is a member of the cysteine-rich scavenger receptor family, highly specific for leukocytes of the mononuclear phagocyte lineage. In vitro, it is induced by glucocorticoids, interleukin-6 (IL-6), and IL-10 and down-regulated by interferon-gamma (IFNgamma), indicating that it has a role in antiinflammatory or other immunomodulatory pathways. We assessed CD163 expression in microenvironments within rheumatoid arthritis (RA) synovium to clarify the relationships among CD4+ T lymphocytes, IFNgamma, and macrophage function in RA. METHODS: Double immunofluorescence and serial immunoenzymatic studies were performed on normal, osteoarthritic, and RA synovium and tonsil with antibodies to CD163, CD45, CD68, CD14, CD3, CD4, CD8, CD19, and IFNgamma. RESULTS: CD163 was observed on all CD14+ cells in synovium and tonsil with the exception of cells within larger T lymphocyte clusters in synovium and within tonsillar follicles. All brightly CD14+ cells in or around vessel walls (interpreted as immigrant monocytes) were CD163+. CD163 labeled fewer cells than did CD68 in synovial intima, but all CD45+ intimal cells were CD163+. CD4+,IFNgamma+ T lymphocytes in RA synovium were chiefly localized within clusters containing CD68+, CD163- cells. CONCLUSION: Within RA synovium, CD163 has major advantages as a macrophage marker and does not appear to be restricted to "mature" macrophages. CD163 discriminates between synovial macrophages and synovial intimal fibroblasts, which also stain positively for CD68 in diseased tissue.

Failed export of the adrenocorticotrophin receptor from the endoplasmic reticulum in non-adrenal cells: evidence in support of a requirement for a specific adrenal accessory factor.

Difficulty in expressing the adrenocorticotrophin (ACTH) receptor (melanocortin 2 receptor; MC2R) after transfection of various MC2R expression vectors has been experienced by many researchers. Reproducible evidence for expression has been obtained only in the Y6/OS3 corticoadrenal cell lines or in cells expressing endogenous melanocortin receptors. In order to determine the cause of this failure of expression we have undertaken the following studies. An MC2R expression plasmid was constructed in which the green fluorescent protein (GFP) coding region had been added to the C-terminus of the mature protein. Transfection of this plasmid into Y6 cells with a cAMP-responsive reporter plasmid demonstrated normal function of this receptor. Imaging of CHO cells expressing MC2R-GFP revealed perinuclear expression, although a cholecystokinin receptor (CCKR)-GFP construct was efficiently expressed at the cell surface. Y6 cells, in contrast, showed cell surface fluorescence after transfection with MC2R-GFP. Several other cell types showed a similar pattern of GFP distribution characteristic of retention in the endoplasmic reticulum. Counterstaining with an anti-KDEL antibody confirmed this location. Co-expression of the MC2R and the CCKR-GFP did not impair CCKR trafficking to the cell surface, implying a receptor-specific impairment to trafficking in the CHO cell which was absent in the Y6 cell.

Transfection of annexin 1 in monocytic cells produces a high degree of spontaneous and stimulated apoptosis associated with caspase-3 activation.

Transfection of the pre-monomyelocytic U937 cell line with a plasmid coding for full-length annexin 1 (ANX1, 347 amino acid) leads to cell death by promoting apoptosis. In addition, over-expression of the N-terminal and the first domain of the protein (144 amino acids, clone ANX1-S), which does not contain the Ca2+ binding sites, gives susceptibility to cell apoptosis following activation by either 5 ng ml(-1) tumour necrosis factor (TNF)-alpha or 1 - 40 microg ml-1 etoposide. This was demonstrated by using the fluorescent labelled annexin V, cell cycle and nuclear staining analyses. Transfection with an empty plasmid (clone CMV) or with a plasmid carrying the cDNA antisense for ANX1 (clone ANX1-AS) did not alter U937 cells to the degree of apoptosis promoted by either stimulant. Treatment of CMV U937 cells with TNF-alpha increased ANX1 mRNA and protein expression in a time-dependent manner, with maximal increases at 3 and 6 h, respectively. Clone ANX1-S showed higher constitutive (more than 2 fold) and activated caspase-3 activity, associated with higher phospholipase A2 (PLA2) activity (in the region of +50 - 100%), whereas expression of cytosolic PLA2 Bax and Bcl-2 were similar in all cell clones, as determined by Western blotting. In conclusion, this study demonstrates a complex regulatory role of cell apoptosis for ANX1, at least with regards to cells of the myelo-monocytic lineage.

Human monocytes express CD163, which is upregulated by IL-10 and identical to p155.

CD163 is a glucocorticoid-inducible member of the scavenger receptor cysteine-rich family of proteins. Previous reports have indicated that CD163 is highly expressed on human macrophages, but found on less than 50% of peripheral blood monocytes. We now show that >99% of all CD14 positive monocytes express CD163 and that monocyte derived dendritic cells express low levels of CD163. We also show that IL-10, like glucocorticoids, induces high CD163 expression on cultured human monocytes. Glucocorticoid induced CD163 expression was not inhibited by anti-IL-10 and was additive with IL-10 treatment, suggesting that glucocorticoids increase CD163 expression by an IL-10 independent mechanism. Other anti-inflammatory cytokines (IL-4 and IL-13) did not increase CD163 expression. In addition, we show that p155 (a previously identified monocyte/macrophage marker of unknown function) shares identity with CD163. Western blots and flow cytometric analysis of HEK 293 cells transfected with the cDNA for CD163 were positive when probed with either mAb RM3/1 (which recognizes CD163) or Mac 2-48 (which defines p155).

Increased apoptosis in U937 cells over-expressing lipocortin 1 (annexin I).

The potential involvement of endogenous lipocortin 1 in the process of cellular apoptosis, particularly in cells of the myelo-monocytic lineage, has been investigated. U937 cells were transfected either with an antisense or a sense DNA for lipocortin 1 and the stable clones 36.4AS clone (20-40% lower lipocortin 1 levels) and 15S (30% higher lipocortin 1 levels) were obtained. Cell apoptosis was induced by incubation with tumor necrosis factor-alpha: optimal responses were observed within a 24 h incubation period at a 5 ng/ml concentration. Apoptosis was assessed both morphologically, by annexin V binding and cell cycle analysis with propidium iodide. Whilst no consistent difference was seen between wild type cells and clone 36.4AS, a higher incidence of apoptosis (ranging from +30% to + 60%) was observed in the 15S clone. Release of arachidonic acid from loaded cells was promoted by 24 h incubation with the cytokine, and a higher degree of release was measured in the 15S clone. These data indicate that endogenous intracellular lipocortin 1 is involved in the promotion of apoptosis in cells of the myelo-monocytic derivation.

Annexin I is stored within gelatinase granules of human neutrophil and mobilized on the cell surface upon adhesion but not phagocytosis.

Annexin I, a member of the calcium- and phospholipid-binding annexin superfamily of proteins, is largely present in human neutrophils. To determine its exact intracellular distribution a combination of flow cytometry, confocal microscopy and electron microscopy analyses were performed on resting human neutrophils as well as on cells which had been activated. In resting neutrophils, annexin I was found to be present in small amounts in the nucleus, in the cytoplasm and partially also associated with the plasma membrane. The cytoplasmic pool of annexin I was predominant, and the protein was co-localized with gelatinase (marker of gelatinase granules), but not with human serum albumin or CD35 (markers of secretory vesicles), or with lysosomes. Electron microscopy showed the presence of annexin I inside the gelatinase granules. Neutrophil adhesion to monolayers of endothelial cells, but not phagocytosis of particles of opsonized zymosan, provoked an intense mobilization of annexin I, with a marked externalization on the outer leaflet of the plasma membrane. Remaining intracellular annexin I was also found in proximity of the plasma membrane. These results provide a novel mechanism for annexin I secretion from human neutrophils, which is via a degranulation event involving gelatinase granules.

The inhibitory effect of dexamethasone on lymphocyte adhesion molecule expression and intercellular aggregation is not mediated by lipocortin 1.

Glucocorticoids exert their anti-inflammatory activity through multiple pathways which include the inhibition of cell adhesion events. The glucocorticoid-induced protein lipocortin 1 (LC1) has reported anti-inflammatory properties and has been proposed as a putative mediator of the anti-inflammatory effects of glucocorticoids. The role of LC1 in mediating the glucocorticoid inhibition of lymphocyte adhesion and cell adhesion molecule (CAM) expression was investigated in vitro using a microaggregation assay, flow cytometry and confocal microscopy. Lymphocytes stimulated for 96 h with plastic-bound OKT3 antibody showed significant increases in LFA-1 and CD2 expression. Dexamethasone (DEX; 10(-6) M) inhibited this increase but the neutralizing anti-LC1 MoAb 1A (5 microg/ml) failed to reverse the DEX effect; neither was purified human LC1 (50 x 10(-9) M) able to inhibit CAM expression. The biological activity of the LC1 was confirmed by its ability to suppress monocyte phagocytosis and respiratory burst in response to bovine serum albumin (BSA)-anti-BSA complexes. OKT3 stimulation of cultured mononuclear cells resulted in intercellular aggregation, scored microscopically using a visual index. This aggregation was completely reversed by 10-6 M DEX but unaffected by LC1 (50 x 10(-9) M). Significant intracellular expression of lymphocyte LC1 was observed using the anti-LC1 MoAb 1B in saponin-permeabilized cells. Distribution of LC1 had a diffuse, cytoplasmic pattern. LC1 expression was reduced following 3 h treatment with 10(-6) M DEX. These findings indicate that the DEX effects on lymphocyte adhesion and CAM expression are not mediated by LC1. Thus the reported in vivo effects of LC1 on leucocyte adhesion and transmigration probably occur through functional/conformation changes of surface CAM, rather than by alteration in expression.

Differential modulation of annexin I binding sites on monocytes and neutrophils.

Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN). These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.

Novel pathways for glucocorticoid effects on neutrophils in chronic inflammation.

Neutrophils have been implicated in mediating much of the tissue damage associated with chronic inflammatory diseases such as rheumatoid arthritis, where they are involved in destruction of both cartilage and bone. Glucocorticoids are powerful anti-inflammatory agents, often used in the treatment of this autoimmune disease. They exert significant inhibitory effects on neutrophil activation and functions, such as chemotaxis, adhesion, transmigration, apoptosis, oxidative burst, and phagocytosis. The mechanisms by which glucocorticoids exert these effects on neutrophils are unclear. Evidence from studies of inflammation in human subjects and animal models suggests that annexin-I an endogenous, glucocorticoid-induced protein also known as lipocortin-1, has a pivotal role in modulating neutrophil activation, transmigratory, and phagocytic functions. Furthermore, we present evidence for altered neutrophil functions in rheumatoid arthritis that correspond to a significantly reduced capacity of these cells to bind annexin-I. A proposed novel pathway for glucocorticoid actions on neutrophils involving annexin-I could explain the development of chronic neutrophil activation in diseases such as rheumatoid arthritis.

Mobilizing lipocortin 1 in adherent human leukocytes downregulates their transmigration.

Polymorphonuclear leukocyte (PMN) migration into sites of inflammation is fundamental to the host defense response. Activation of endothelial cells and PMNs increases the expression or activation of adhesion molecules, culminating in rolling and subsequent adherence of these cells to the vascular wall. Further activation of adherent PMNs, possibly by endothelial cell ligands, leads, within a few minutes, to extravasation itself. This process is not clearly understood, but adhesion molecules or related proteins, as well as endogenous chemokines, may play an important role. The anti-inflammatory glucocorticoids delay extravasation, which implies that an inhibitory regulatory system exists. Resting PMNs contain abundant cytoplasmic lipocortin 1 (LC1, also called annexin I)', and the activity profile of this protein suggests that it could reduce PMN responsiveness. To investigate this we have assessed neutrophil transmigration both in vivo and in vitro and examined the content and subcellular distribution of LC1 in PMNs by fluorescence-activated cell-sorting (FACS) analysis, western blotting and confocal microscopy. We report that LC1 is mobilized and externalized following PMN adhesion to endothelial monolayers in vitro or to venular endothelium in vivo and that the end point of this process is a negative regulation of PMN transendothelial passage.

Evidence for specific annexin I-binding proteins on human monocytes.

Recombinant human annexin I and a monoclonal antibody specific for this protein (mAb 1B) were used to investigate surface binding of this member of the annexin family of proteins to peripheral blood monocytes. Flow cytometric analysis demonstrated trypsin-sensitive, saturable binding of annexin I to human peripheral blood monocytes but not to admixed lymphocytes. A monoclonal antibody that blocks the anti-phospholipase activity of annexin I also blocked its binding to monocytes. These findings suggest the presence of specific binding sites on monocytes. Furthermore, surface iodination, immunoprecipitation and SDS/PAGE analysis were used to identify two annexin I-binding proteins on the surface of monocytes with molecular masses of 15 kDa and 18 kDa respectively. The identification and characterization of these annexin I-binding molecules should help us to better understand the specific interactions of annexin I with monocytes that lead to down-regulation of pro-inflammatory cell functions.

Differential distribution of annexins-I, -II, -IV, and -VI in synovium.

OBJECTIVES: To examine the distribution of four annexins in non-inflamed rheumatoid arthritic and osteoarthritic synovial tissue. METHODS: Frozen sections were stained with monoclonal antibodies (MAb) specific for annexins-I, -II, -IV, and -VI, and for cell lineage related markers including CD68 and CD14 (macrophages), prolyl hydroxylase (fibroblasts), and CD3 (T cells). RESULTS: Each of the annexins was present in synovial tissues in significant amounts in the three groups studied. Annexin-I was predominantly found within the synovial lining layer and double labelling showed it to be present predominantly in cells of the macrophage lineage. In rheumatoid specimens there was increased staining within the lining layer, perivascularly and on macrophages within the tissue stroma. Annexin-II was present in a distribution similar to that of annexin-I, but with more prominent perivascular staining. Annexins-IV and -VI were seen chiefly in association with areas of lymphocyte infiltration in rheumatoid tissue, whereas annexins-I and -II were absent from these areas. Endothelial cells stained weakly positive for annexins-I and -II, and more strongly for -IV and -VI. CONCLUSIONS: This study demonstrates that annexins (particularly annexin-I, a putative mediator of the anti-inflammatory activities of glucocorticoids) are abundant in rheumatoid and non-rheumatoid synovial tissue, annexins-IV and -VI having a distribution distinct from that of -I and -II.

Potential role of Clara cell protein, an endogenous phospholipase A2 inhibitor, in acute lung injury.

It is now recognized that epithelial cells lining airways and alveoli are capable of releasing various mediators, which have the potential to modulate local inflammatory reactions. The amount of the 16 kDa Clara cell protein (CC16), an inhibitor of phospholipase A2 activity produced by pulmonary epithelial cells, was measured by means of a sensitive immunoassay in the unconcentrated bronchoalveolar lavage fluid (BALF) of 13 control subjects, and in patients with acute lung injury (14 with the full-blown adult respiratory distress syndrome (ARDS); 21 after standard cardiopulmonary bypass surgery, a known risk factor for ARDS). The level of CC16 was compared with other markers of inflammation with a wide range of molecular weights: albumin (nephelometry); total protein (spectrophotometry); beta 2-microglobulin (latex immunoassay); cystatin C (latex immunoassay); alpha 1-antitrypsin (immunoradiometry), and lipocortin-1 (enzyme-linked immunosorbent assay (ELISA)). The Clara cell protein (CC16) was detectable in all BALF, and significantly higher levels of this protein were observed in BALF from patients with acute lung injury. Changes in BALF Clara cell protein levels differed from those of alpha 2-macroglobulin and the natural phospholipase inhibitor lipocortin-1. Alpha 2-macroglobulin levels were not significantly enhanced in patients at risk for ARDS, but were increased in patients with ARDS; whereas, lipocortin 1 levels were not elevated in either group. Pretreatment of patients at risk for ARDS with high dose methylprednisolone did not alter the amount of Clara cell protein recovered in BALF. The mean CC16 level in BALF from patients with ARDS who died was significantly lower than from those who survived. The data presented in this study suggest that pulmonary epithelial cells secrete a natural anti-inflammatory protein during acute lung injury, which might have a protective and immunosuppressive role.

Lipocortin-1 autoantibody concentration in children with inflammatory bowel disease.

BACKGROUND: Corticosteroids are widely used to treat children with inflammatory bowel disease although the response is variable, side-effects are common, and many patients develop a partial or complete steroid resistance. The mechanism underlying these phenomena are unclear. Corticosteroids mediate some of their actions through lipocortin-1, and the induction of autoantibodies to lipocortin has been proposed as a possible mechanism by which steroid efficacy is suboptimal in vivo. PATIENTS AND METHODS: We have measured serum lipocortin-1 antibody concentration by ELISA in 38 children with Crohn's disease, 12 with ulcerative colitis and in 15 controls. RESULTS: IgG and IgA anti-lipocortin-1 antibody levels were higher in the Crohn's group than in the ulcerative colitis or control groups. Elevated concentrations did not relate to disease activity, history of steroid therapy or steroid-responsiveness. Lipocortin IgM antibody status was similar in all three groups. CONCLUSION: It is therefore unlikely that serum antibodies to lipocortin-1 have a role in the development of steroid-resistance in children with inflammatory bowel disease.

Detection of intracellular lipocortin 1 in human leukocyte subsets.

Lipocortin 1, a putative mediator of the anti-inflammatory actions of glucocorticoids, is present intracellularly in a variety of tissues including human peripheral blood leukocytes. We investigated the presence of lipocortin 1 in human leukocyte subsets using permeabilization flow cytometry. Constitutive lipocortin 1 was detected in U937 myelomonocytic leukemia cells, and lipocortin 1 was increased by treatment with PMA or PMA+IFN-gamma (P < 0.05) but not by dexamethasone. Lipocortin 1 was present in all leukocyte subsets except B lymphocytes (CD19/20+, P < 0.001). Lipocortin 1 content was maximal in monocytes and polymorphonuclear neutrophils and least in lymphocytes (P < 0.001). Monocyte lipocortin 1 was strongly associated with surface expression of CD14 and HLA-DR. Among non-B lymphocytes, a range of lipocortin 1 fluorescence was observed. Lipocortin 1 fluorescence was greatest in natural killer cells (CD56+, P < 0.001) and CD57+ cells, but T cell subset markers did not otherwise discriminate variations in lipocortin 1. Induction of lymphocyte proliferation by PHA, anti-CD3, Con A, superantigen, and SAC was not associated with significant shifts in lipocortin 1 content. Dexamethasone (10(-10)-10(-6) M) did not induce increases in PB leukocyte lipocortin 1. We conclude that lipocortin 1 content in human leukocytes varies significantly among phenotypic subsets. This has significance for the investigation of inflammatory disease where certain cell types predominate.

Abnormal hypothalamic-pituitary-adrenal axis function in rheumatoid arthritis. Effects of nonsteroidal antiinflammatory drugs and water immersion.

OBJECTIVE: To investigate the effects of nonsteroidal antiinflammatory drug (NSAID) therapy and water immersion on hypothalamic-pituitary-adrenal (HPA) axis function in rheumatoid arthritis (RA). METHODS: Plasma levels of adrenocorticotropic hormone (ACTH) and serum and urine levels of cortisol were compared in untreated RA patients, NSAID-treated RA patients, and healthy control subjects. RESULTS: ACTH levels were significantly higher in untreated RA patients (mean +/- SEM integrated area 11,377 +/- 5,246 hours ng/liter) than in NSAID-treated RA patients (2,285 +/- 388 hours ng/liter) or healthy controls (1,845 +/- 35.5 hours ng/liter) (P < 0.001). Serum and urine cortisol levels were not significantly different between groups. Two-hour head-out water immersion had no effect. CONCLUSION: Elevated ACTH levels without hypercortisolemia occur in untreated RA. NSAID therapy alters HPA axis response, but immersion has no effect.

Circulating group II phospholipase A2 activity and antilipocortin antibodies in systemic lupus erythematosus. Correlative study with disease activity.

OBJECTIVE: Lipocortin (LC) and phospholipase A2 (PLA2) are involved in phospholipid metabolism, and on the cellular level LC seems to be an antagonist of PLA2. Since anti-LC1 autoantibodies were found in systemic lupus erythematosus (SLE), we undertook a study of the relationship between PLA2, anti-LC1, and disease activity in a large group of patients with SLE. METHODS: Sera from 81 patients with SLE were tested for the activity of extracellular PLA2 and the presence and level of antilipocortin 1 [anti-LC1 (IgM) and anti-LC1 (IgG)] antibodies. Both were compared to SLE activity. RESULTS: Mean PLA2 activity was 4.6-fold higher in patients with SLE than in healthy controls (707 +/- 219 vs 154 +/- 6 u/ml, p < 0.01). PLA2 activity correlated significantly with PLA2 immunoreactivity as estimated by an ELISA method using monoclonal antibodies against "synovial type" PLA2 (n = 21, r = 0.984, p < 0.001). Anti-LC1 IgM and IgG antibody levels were significantly higher in SLE than in healthy individuals [anti-LC1 (IgM) 54.5 +/- 4.6 vs 22.6 +/- 2.3 EU/ml, p < 0.001 and anti-LC1 (IgG) 54.3 +/- 3.4 vs 22.9 +/- 2.3 EU/ml, p < 0.001]. There was no correlation between PLA2 activity and anti-LC1 antibody titers. Elevated levels of PLA2 [> normal mean + 2 SD (i.e., > 300 u/ml)] were found in 41/81 patients with SLE. Anti-LC1 antibody titers were high (> 64 EU/ml) in 23/41 patients; 14/40 patients with SLE with normal PLA2 (< 300 u/ml) also had higher titers of anti-LC1 antibodies. PLA2 activity was significantly associated with the presence of synovitis, being markedly increased in 11/12 patients. Mean PLA2 in this group of patients (1593 +/- 957 u/ml) was significantly higher (p < 0.001) than that (553 +/- 188 u/ml) in the group of 69 patients with SLE without synovitis. CONCLUSIONS: There was no correlation of PLA2 activity with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) or the Lupus Activity Criteria Count (LACC). Circulating PLA2 activity in SLE correlated only with active synovitis. There was no correlation of anti-LC1 titers with duration of the disease, age, steroid dosage, SLEDAI, or LACC or any individual clinical or laboratory variable included in the assessment of SLEDAI and LACC.

Impaired glucocorticoid induction of mononuclear leukocyte lipocortin-1 in rheumatoid arthritis.

OBJECTIVE: To investigate leukocyte lipocortin-1 production in rheumatoid arthritis (RA). METHODS: Eight control and 8 RA subjects received 100 mg hydrocortisone intravenously. Leukocyte lipocortin-1 was measured by enzyme-linked immunosorbent assay. RESULTS: Hydrocortisone induced significant increases in lipocortin-1 production by control mononuclear cells (MNC) (P = 0.006 versus baseline) but not by RA MNC (P = 0.44 versus baseline). Peak lipocortin-1 levels in control MNC were significantly higher than those in RA MNC (P = 0.014). CONCLUSION: These results indicate that glucocorticoid-induced MNC lipocortin-1 production is impaired in RA.