Immunoreactants in rheumatoid synovial effusions.

Measurements were made of the IgG in synovial fluid which is synthesized by the intact rheumatoid synovium. In five rheumatoid subjects 12-24% of the IgG present in their synovial fluid was derived from local production. No evidence for local production was found in patients with degenerative arthritis or Reiter's syndrome. It was estimated that as much as 95 mg of IgG was produced by the synovium of a single knee joint daily. Rheumatoid inflammation is associated with the presence of large numbers of white blood cells, predominantly polymorphonuclear, in the articular cavity. Each day as many as one billion white cells enter the articular cavity to participate in this inflammatory response. Factors causing the directed migration of polymorphonuclear leukocytes were found in the majority of rheumatoid effusions studied. The chemotactic activity is, in large part, related to the fifth (C5) and sixth (C6) components of human complement. Physical-chemical techniques indicate that the activity is attributable to C 567 and C5a, a cleavage product of C5. In addition to the presence of preformed chemotactic factors, more than half of the rheumatoid fluids contain an enzyme capable of generating chemotactic activity from the fifth component (C5) of human complement. This information supports the concept that rheumatoid joint inflammation is an example of immune complex disease in which a significant proportion of the immunoreactants are derived from local production.

Characterization of the killer cell generated in the autologous mixed leukocyte reaction.

Cytotoxic cells are produced in an autologous mixed leukocyte reaction (AMLR). At 1 wk in culture the AMLR killers are mainly IgG Fc- cells and can kill autologous lymphoblastoid cell lines and Raji and Daudi targets that are usually resistant to natural killer cell (NK) lysis. To define the phenotype of these cells, we have used complement (C')-mediated lysis with monoclonal antibodies (MAb). AMLR killer activity was virtually eliminated by treatment with C' and 9.6 or 4F2, but the cytotoxic cells did not express NK-specific antigens, OKM1 and Leu-7, nor cytolytic T lymphocyte-specific antigens, 9.3 and OKT8. None of the 10 MAb used could significantly block cytotoxicity at the final concentration of 1.5 mcg/ml which is generally sufficient to inhibit CTL. The majority of cells at 1 wk in AMLR cultures stained with T cell activation antigens Ia and 4F2; AMLR killing was proportional to the percentage of 4F2+ cells but unrelated to the expression of Ia antigen.

Rabbit anaphylactic antibody.

Antibody capable of sensitizing rabbit skin for passive cutaneous anaphylaxis is produced in the rabbit as early as 6 to 7 days following antigenic stimulation. It reaches peak activity around the 9th day and is gone by the 3rd wk. The antibody is heat labile, sensitive to treatment with mercaptoethanol, non-precipitating and does not fix complement. In order to demonstrate PCA activity a latent period is required of from 48 to 72 hr after introduction of the antibody into the rabbit's skin; the activity can persist for at least 17 days. It has a faster electrophoretic mobility than rabbit gammaG-globulin, and is eluted somewhat earlier than gammaG-globulin from Sephadex G-200, although distinctly after gammaM-globulin. No relationship was demonstrated between the rabbit PCA activity and the hemagglutinating activity found in the same sera. The rabbit anaphylactic antibody differs in almost all properties studies from the rabbit 7S antibody capable of sensitizing guinea pigs for PCA which arises at the same time. This latter antibody found early in immunization had properties which were indistinguishable from those described for the rabbit 7S antibody giving PCA in the guinea pig found in late hyperimmune sera.

Rabbit homocytotropic antibody. A unique rabbit immunoglobulin analogous to human IgE.

Rabbits immunized with egg albumin produce a homocytotropic antibody. The antibody is identified by its ability to produce passive anaphylaxis in rabbit skin. The time of appearance of this antibody, its persistence and recall after booster injections depends, in part, on the route of immunization and the adjuvant employed. The physicochemical characteristics of the homocytotropic antibody obtained was similar regardless of the immunization schedule used. The anaphylactic activity of these antisera showed some heterogeneity when chromatographed on diethylaminoethyl (DEAE)-cellulose, but all fractions were inactivated by heating and absorption with a specific antisera. The anaphylactic activity could be separated from rabbit IgG and IgA, and was not blocked by absorption with antisera specific for these classes of immunoglobulins. Anaphylactic activity was completely removed by absorption with a specific antiserum which did not react with any of the known rabbit immunoglobulins. The passive cutaneous anaphylaxis titer of a rabbit serum containing homocytotropic antibody was reduced by 50% after absorption with an antisera (anti-FcND) specific for human IgE. On the basis of these distinctive physicochemical characteristics, it is concluded that rabbit homocytotropic antibody represents a unique class of rabbit immunoglobulin, analogous to human IgE.

Cytokines in chronic inflammatory arthritis. IV. Granulocyte/macrophage colony-stimulating factor-mediated induction of class II MHC antigen on human monocytes: a possible role in rheumatoid arthritis.

Granulocyte/macrophage CSF (GM-CSF) has recently been identified in rheumatoid arthritis (RA) synovial effusions. To study a potential role for GM-CSF and other cytokines on the induction of HLA-DR expression on monocytes and synovial macrophages, we analyzed the relative ability of recombinant human cytokines to induce the surface expression of class II MHC antigens on normal peripheral blood monocytes by FACS analysis. GM-CSF (800 U/ml) (mean fluorescence channel 2.54 +/- 0.33 times the control, p less than 0.001) and IFN-gamma (100 U/ml) (5.14 +/- 0.60, p less than 0.001) were the most potent inducers of HLA-DR. TNF-alpha and IL-4 also increased HLA-DR expression, although to a lesser degree [1.31 +/- 0.06 (p less than 0.02) and 1.20 +/- 0.03 (p less than 0.01), respectively]. IL-1 (40 U/ml), IL-2 (10 ng/ml), IL-3 (50 U/ml), IL-6 (100 U/ml), and CSF-1 (1,000 U/ml) did not affect surface HLA-DR density. GM-CSF also increased HLA-DR mRNA expression and surface HLA-DQ expression, but decreased CD14 (a monocyte/macrophage antigen) expression. The effect of GM-CSF on HLA-DR was not mediated by the generation of IFN-gamma in vitro because it was not blocked by anti-IFN-gamma mAb. GM-CSF was additive with IL-4 and low amounts (less than 3 U/ml) of IFN-gamma and synergistic with TNF-alpha. Because we have recently reported that supernatants of cultured RA synovial cells produce a non-IFN-gamma factor that induces HLA-DR on monocytes, we then attempted to neutralize this factor with specific anti-GM-CSF mAb. Four separate synovial tissue supernatants were studied, and the antibody neutralized the HLA-DR-inducing factor in each (p less than 0.01).

Analysis of the defects responsible for the impaired regulation of Epstein-Barr virus-induced B cell proliferation by rheumatoid arthritis lymphocytes. I. Diminished gamma interferon production in response to autologous stimulation.

T cells of patients with rheumatoid arthritis (RA) do not control the rate of B lymphoblast transformation induced by Epstein-Barr virus (EBV) as efficiently as T cells from healthy individuals; thus, lymphoblast cell lines are established more readily in RA lymphocytes in vitro after EBV infection. In the present experiments, we have asked whether this T cell regulation can be reproduced by lymphocytes. We found that normal T cells, activated in allogeneic or autologous mixed leukocyte reactions (MLR), produce lymphokines that inhibit in vitro EBV-induced B cell proliferation. Allogeneic MLR supernatants inhibited EBV-induced DNA synthesis 62 +/- 4% (mean +/- SE) at 10 d post-infection, whereas autologous MLR supernatants suppressed it 50 +/- 3%. RA T cell supernatants produced in an allogeneic MLR suppressed as well as normal T cell supernatants (64 +/- 5% inhibition). In contrast, supernatants from RA autologous MLR had little inhibitory activity. EBV-induced DNA synthesis at 10 d was reduced only 8 +/- 3%, compared with the 50 +/- 3% suppressive activity of normal autologous MLR supernatants. The magnitude of the proliferative responses in the autologous MLR regenerating the lymphokines was similar in the normal and RA populations. After depletion of adherent cells from the RA auto-MLR stimulators, supernatant inhibitory activities increased to normal levels (from 11 +/- 6 [SE] to 52 +/- 6% [SE]). The inhibitory factor involved in the regulation of in vitro EBV infection is a protein with a molecular weight of approximately 50,000. Its activity is eliminated by hearing at 56 degrees C and by exposure to acid at pH 2. The inhibitory activity is blocked by mixing the MLR supernatants with a polyvalent antisera or monoclonal antibodies specific for human gamma interferon. Gamma interferon produced by activating T cells in allo- or auto-MLR can reproduce T cell-mediated regulation of EBV-induced B cell proliferation, and the failure of RA auto-MLR to generate that lymphokine parallels the defective T cell regulation of EBV-induced B cell proliferation characteristic of RA lymphoid cells.

Cytokines in chronic inflammatory arthritis. I. Failure to detect T cell lymphokines (interleukin 2 and interleukin 3) and presence of macrophage colony-stimulating factor (CSF-1) and a novel mast cell growth factor in rheumatoid synovitis.

Because previous studies showed low levels of IFN-gamma in rheumatoid arthritis (RA) synovial fluid (SF) and synovial tissue (ST) explant supernatants, we assayed RA SF and ST for IL-2 and IL-3-like activity. Using an IL-2 dependent murine CTLL line, 6 of 14 RA SF caused increased thymidine uptake (greater than three times control). The activity was distinct from IL-2 because it was not blocked by antibody to IL-2-R. In addition, IL-2 was not detected (less than 50 pg/ml) in 16 joint samples using an ELISA. Multi-colony-stimulating factor (CSF) activity was measured using two assays that can detect murine IL-3 (mast cell proliferation, and bone marrow CSF). In the mast cell assay, [3H]TdR uptake was 493 +/- 67 cpm for medium, 2,910 +/- 329 cpm in the presence of RA SF (p less than 0.001), 1,246 +/- 156 cpm in the presence of SF from patients with seronegative spondyloarthropathies (p less than 0.001), and 736 +/- 100 cpm in the presence of osteoarthritis SF (p greater than 0.1). In the CSF assay, four of five RA SF and five of five RA ST induced colony formation from bone marrow nonadherent cells. Macrophage colonies were most common, although mixed colonies and granulocytes were occasionally observed. The multi-CSF activity in RA is not due to IL-3 since human rIL-3 was not active in either murine assay, and IL-3 mRNA was not detected in RA synovium. Sephadex column chromatography of RA SF revealed that the mast cell growth factor (approximately 6 x 10(3) mol wt) and the CSF (approximately 40 and 100 x 10(3) mol wt) are distinct. The colony-stimulating aspect of the "IL-3-like" activity in RA SF is likely due to CSF-1 because it is the appropriate mol wt and because the activity was neutralized by specific anti-CSF-1 antibody. Finally, an RIA detected 1.6-25 ng/ml of CSF-1 in RA SF and ST and CSF-1 mRNA was detected in four of five RA synovial tissue samples tested.

Breakdown products of C 3 in human synovial fluids.

Activation of the complement sequence results in conversion of the third component of complement (C'3) to an inactive product (C'3i) and the elaboration of additional fragments of smaller molecular weights and faster electrophoretic mobilities. Immunoelectrophoretic analysis of fresh synovial fluids with an anti-human C'3 antiserum disclosed in some a variable degree of conversion of C'3 to C'3i, but a more striking finding was an additional line in the alpha-globulin region. This faster migrating protein gave a reaction of partial identity with C'3/C'3i. With this antiserum a similar pattern developed when fresh human serum was incubated with immune complexes, or aggregated gamma-globulin. The same breakdown product of C'3 was obtained by treatment of fresh human serum with Zymosan, ammonium, hydrazine, agar, or dextran. Heating serum at 56 degrees C for 1 hr destroys the breakdown product; aging of serum produces it. Breakdown products of C'3 were looked for in 49 synovial fluids from patients with a variety of joint diseases. A significant correlation was found between the demonstration of the fast migrating breakdown product of C'3 and the diagnosis of rheumatoid arthritis and the presence of rheumatoid factor. A similar immunoelectrophoretic pattern was not found in the serum of any of the patients studied. When human gamma-globulin, which has been reduced and alkylated, is heat aggregated it loses the ability to fix human complement but still reacts with rheumatoid factor. Addition of reduced, aggregated gamma-globulin to fresh normal human serum produced no conversion of C'3, but when incubated with serum containing a high titer of rheumatoid factor, there was conversion of C'3 and the appearance of a breakdown product. Quantitative complement fixation studies with fresh serum from normal subjects and patients with rheumatoid arthritis disclosed complement fixation by reduced, aggregated gamma-globulin. The per cent of complement fixation was proportional to the titer of rheumatoid factor present in the test serum. These findings were interpreted as showing that rheumatoid factor can fix complement.The possibility is discussed that the presence of breakdown products of C'3 in the synovial effusions of most patients with seropositive rheumatoid arthritis and the ability of rheumatoid factor to fix complement are related phenomena.

Dendritic cell-lymphocyte clusters that form spontaneously in rheumatoid arthritis synovial effusions differ from clusters formed in human mixed leukocyte reactions.

Lymphocytes cluster about dendritic cells (DC) spontaneously in 48 h cultures of rheumatoid arthritis synovial fluid (RA SF) mononuclear cells and in peripheral blood autologous or allogeneic mixed leukocyte reactions. In the latter case, the clusters are predominantly CD4+ T cells (T4/T8 greater than 5) and with time progress in blastic cells that express IL-2 (Tac) and/or transferrin (T9) receptors. In contrast, the clusters in RA SF cultures have a T4/T8 ratio of less than 1 and a majority of the T8 cells coexpress the Leu 7 marker. T cells in these clusters remain inert and with time the clusters disintegrate. Addition of IL-1, IL-2, or IFN-gamma alone or in combination had no effect on RA SF clusters but T cells became blastic when exposed to 10% RA SF. Mixing experiments using RA SF DC with normal T cells and RA T cells with normal DC show that both RA SF DC and T cells are immunofunctional. In addition, clusters of RA SF from a patient with active tuberculosis proliferated vigorously to PPD. Therefore, the unique RA SF cluster profile may reflect the memory nature of the RA SF T cells resulting in a paucity of T cells that are responsive to autologous stimulation. However, an immunosuppressive role for the double-labeled (CD8 and Leu 7) cells has not been excluded.

Brain-reactive lymphocytotoxic antibodies in the serum of patients with systemic lupus erythematosus.

Homogenized tissue from the frontal cortex of normal human brains obtained at postmortem examination was used to absorb lymphocytotoxic antibody from the serum of six patients with systemic lupus erythematosus (SLE). Four absorptions of all of the SLE sera with equal volumes of homogenized brain tissue at 4 degrees C depleted their cytotoxic capacity more than 90%. Three of the six sera, however, retained some lymphocytotoxicity despite extensive brain absorption. Absorbed lymphocytotoxic antibodies were eluted from brain tissue absorbents at 37 degrees C. Cytotoxicity of the brain eluates was blocked by antibodies to human IgM (mu-chain specific) but not anti-IgG. The unabsorbed SLE sera, brain-absorbed sera, and brain eluates were equally cytotoxic to T (thymus-derived) and B (bone marrow-derived) cells fractionated from normal human peripheral blood lymphocytes. Thus, the lymphocytotoxic antibodies in SLE serum exhibit no preference for circulating human T cells. An analysis of the clinical records of 40 patients with SLE whose serum cytotoxic capacity had been determined revealed that circulating lymphocytotoxicity is greater in sera of patients with central nervous system (CNS) manifestations than in other SLE patients. This observation suggests a possible role for brain-reactive lymphocytotoxic antibodies in the development of CNS disease in SLE.

Complement-derived leukotactic factors in inflammatory synovial fluids of humans.

A large per cent of rheumatoid synovial fluids contain chemotactic activity for rabbit granulocytes (neutrophilic). The chemotactic activity is, in large part, related to the fifth (C5) and sixth (C6) components of human complement; a combination of physical-chemical techniques indicates the activity to be attributable to C567 and C5a, a cleavage product of C5. Many rheumatoid synovial fluids contain a C5-cleaving enzyme which, on the basis of substrate specificity and susceptibility to inhibitors, is very similar to an enzyme extractable from lysosomal granules of human and rabbit granulocytes. Inflammatory nonrheumatoid synovial fluids contain chemotactic activity that is related to cleavage products (C3a) of the third component of human complement (C3). Also found in these fluids is a C3-cleaving enzyme capable of producing C3a. Of the other synovial fluids examined, lupus fluids were remarkable by their total lack of chemotactic activity. These findings record for the first time the presence of complement-derived chemotactic factors in pathological human fluids.

Apoptosis in rheumatoid arthritis synovium.

RA synovial tissue (ST) was studied to determine if and where apoptosis occurs in situ. Genomic DNA was extracted from 5 RA and 1 osteoarthritis ST samples. Agarose gel electrophoresis demonstrated DNA ladders characteristic for apoptosis from each tissue. In situ and labeling (ISEL) was used to identify DNA strand breaks consistent with apoptosis in frozen sections. 12 RA and 4 osteoarthritis ST were studied by ISEL and all were positive, but only 2 of 4 normal tissues were positive. The primary location of apopotic cells was the synovial lining. Some sublining cells were also positive, but lymphoid aggregate staining was conspicuously absent. Immunohistochemistry and ISEL were combined and showed that the lining cells with DNA strand breaks were mainly macrophages, although some fibroblastlike cells were also labeled. Sublining cells with fragmented DNA included macrophages and fibroblasts, but T cells in lymphoid aggregates, which expressed large amounts of bcl-2, were spared. DNA strand breaks in cultured fibroblastlike synoviocytes was assessed using ISEL. Apoptosis could be induced by actinomycin D, anti-fas antibody, IL-1, and TNF-alpha but not by IFN-gamma. Fas expression was also detected on fibroblast-like synoviocytes using flow cytometry. Therefore, DNA strand breaks occur in synovium of patients with arthritis. Cytokines regulate this process, and the cytokine profile in RA (high IL-1/TNF; low IFN-gamma) along with local oxidant injury might favor induction of apoptosis.

Cytokines in chronic inflammatory arthritis. V. Mutual antagonism between interferon-gamma and tumor necrosis factor-alpha on HLA-DR expression, proliferation, collagenase production, and granulocyte macrophage colony-stimulating factor production by rheumatoid arthritis synoviocytes.

The effects of a broad array of cytokines, individually and in combination, were determined on separate functions (proliferation, collagenase production, and granulocyte macrophage colony-stimulating factor [GM-CSF] production) and phenotype (expression of class II MHC antigens) of cultured fibroblast-like RA synoviocytes. The following recombinant cytokines were used: IL-1 beta, IL-2, IL-3, IL-4, IFN-gamma, tumor necrosis factor (TNF)-alpha, GM-CSF, and macrophage colony-stimulating factor (M-CSF). Only IFN-gamma induced HLA-DR (but not HLA-DQ) expression. TNF-alpha inhibited IFN-gamma-mediated HLA-DR expression (46.7 +/- 4.1% inhibition) and HLA-DR mRNA accumulation. This inhibitory effect was also observed in osteoarthritis synoviocytes. Only TNF-alpha and IL-1 increased synoviocyte proliferation (stimulation index 3.60 +/- 1.03 and 2.31 +/- 0.46, respectively). IFN-gamma (but none of the other cytokines) inhibited TNF-alpha-induced proliferation (70 +/- 14% inhibition) without affecting the activity of IL-1. Only IL-1 beta and TNF-alpha induced collagenase production (from less than 0.10 U/ml to 1.10 +/- 0.15 and 0.72 +/- 0.24, respectively). IFN-gamma decreased TNF-alpha-mediated collagenase production (69 +/- 19% inhibition) and GM-CSF production but had no effect on the action of IL-1. These data demonstrate mutual antagonism between IFN-gamma and TNF-alpha on fibroblast-like synoviocytes and suggest a novel homeostatic control mechanism that might be defective in RA where very little IFN-gamma is produced.

Cytokines in chronic inflammatory arthritis. II. Granulocyte-macrophage colony-stimulating factor in rheumatoid synovial effusions.

A liquid culture technique was used to study 23 synovial fluids (SF) (21 from inflammatory joint diseases and 2 noninflammatory SF) and supernatants of two cultured rheumatoid arthritis (RA) synovial tissues for colony-stimulating factor (CSF). The proliferative responses of human peripheral blood macrophage-depleted non-T cells treated with synovial fluids, supernatants of synovial tissue explants, and recombinant granulocyte-macrophage (rGM)-CSF were compared. Aggregates of cells that formed in long-term cultures (15 d) were similar for each applied agent and consisted of macrophages, eosinophils, and large blasts. Tritiated thymidine incorporation was proportional to the concentration of rGM-CSF and was accompanied by an increase in number and size of cellular aggregates formed in the cultures. CSF activity was observed in inflammatory SF, with tritiated thymidine uptake of 3,501 +/- 1,140 cpm in the presence of RA samples (n = 15) compared to 1,985 +/- 628 for non-RA inflammatory SF (n = 7) (P less than 0.05) and 583 +/- 525 for medium (n = 6) (P less than 0.01). The proliferative response to RA SF was often more apparent when the samples were diluted, because at higher concentrations the RA SF was inhibitory. Two RA SF were fractionated by Sephadex G100 column chromatography; low levels of CSF activity were detected in fractions corresponding to Mr of 70-100 kD, but the major CSF activity was found in the 20-24-kD fractions. A polyclonal rabbit anti-GM-CSF antibody eliminated the stimulating activity from both rGM-CSF and RA SF. Finally, a specific RIA identified significant levels of GM-CSF (40-140 U/ml) in the culture supernatants of 3 additional RA synovial tissues. These data document the local production of GM-CSF in rheumatoid synovitis and are the first description of this cytokine at a site of disease activity.

Identification of immunostimulatory dendritic cells in the synovial effusions of patients with rheumatoid arthritis.

Dendritic cells in the circulation are leukocytes that are rich in Ia antigens and that actively stimulate T cell replication. We have identified dendritic cells in the joint effusions of patients with rheumatoid arthritis. By phase-contrast and immunofluorescence microscopy, synovial mononuclear cells contained 1-5% dendritic profiles that were rich in HLA-DR and DQ, had small amounts of C3bi receptor, and lacked a battery of monocyte and lymphocyte markers. These dendritic cells could be enriched to 60-80% purity by cytolytic depletion of monocytes and lymphocytes with a group of monoclonal antibodies (MAb) and complement. By transmission electron microscopy, the dendritic cell processes were bulbous in shape and lacked organelles. The cytoplasm had few lysosomes or endocytic vacuoles but contained a well-developed smooth reticulum that was comparable to that previously described in the Ia-rich interdigitating cells of lymphoid tissues. The growth of sodium periodate-modified T lymphocytes was used as a rapid quantitative assay of accessory cell function. Synovial mononuclear cells were some ten times more active than normal blood cells. Treatment with alpha-Ia MAb and complement ablated stimulatory function. In contrast, removal of monocytes (MAb, 3C10) or monocytes and B (MAb, BA-1) plus T (MAb, OKT3, or T101) lymphocytes did not significantly alter total activity, and the function per viable cell increased four- to eightfold. We conclude that rheumatoid arthritis synovial fluids contain cells that are comparable in function, phenotype, and structure to blood dendritic cells, although the frequency (1-5%) is 10 times greater in joints. The reason for their accumulation in the articular cavity is not known, but dendritic cells may be important in perpetuating the joint inflammation characteristic of this disease.

Fibroblast-like synoviocytes support B-cell pseudoemperipolesis via a stromal cell-derived factor-1- and CD106 (VCAM-1)-dependent mechanism.

B-cell accumulation and formation of ectopic germinal centers are characteristic changes in the diseased joints of patients with rheumatoid arthritis (RA). Earlier studies suggested that interactions between B lymphocytes and specialized synovial "nurse-like" cells peculiar to the RA synovium may be responsible for the homing and sustained survival of B cells in the synovium. However, in this study, we found that B cells spontaneously migrate beneath ordinary fibroblast-like synoviocytes (FLSs) and then experience prolonged survival. FLSs isolated from joints of patients with osteoarthritis also supported this activity, termed B-cell pseudoemperipolesis. We found that FLSs constitutively expressed the chemokine stromal cell-derived factor-1 (SDF-1), and that pertussis toxin or antibodies to the SDF-1 receptor (CXCR4) could inhibit B-cell pseudoemperipolesis. However, expression of SDF-1 is not sufficient, as dermal fibroblasts also expressed this chemokine but were unable to support B-cell pseudoemperipolesis unless previously stimulated with IL-4 to express CD106 (VCAM-1), a ligand for the alpha(4)beta(1) integrin, very-late-antigen-4 (VLA-4 or CD49d). Furthermore, mAb's specific for CD49d and CD106, or the synthetic CS1 fibronectin peptide, could inhibit B-cell pseudoemperipolesis. We conclude that ordinary FLSs can support B-cell pseudoemperipolesis via a mechanism dependent upon fibroblast expression of SDF-1 and CD106.

Antiphospholipid antibodies are directed against epitopes of oxidized phospholipids. Recognition of cardiolipin by monoclonal antibodies to epitopes of oxidized low density lipoprotein.

The optimal clinical management of patients with antiphospholipid antibody syndrome (APS) is uncertain because of a lack of an underlying hypothesis to explain why antiphospholipid autoantibodies (aPL) form to such ubiquitous compounds as phospholipids (PL). In this paper, we demonstrate that many, if not most, aPL are actually directed at neoepitopes of oxidized PL, or neoepitopes generated by adduct formation between breakdown products of oxidized PL and associated proteins. Each cardiolipin (CL) molecule contains four unsaturated fatty acids and is highly susceptible to oxidation, particularly upon exposure to air. Yet, standard anticardiolipin antibodies (aCL) immunoassays routinely bind CL to microtiter wells by evaporation of the ethanol solvent overnight at 4 degrees C. Using a variety of techniques, we demonstrated that rapid oxidation occurs when CL is plated and exposed to air. Sera from apo E-deficient mice, which have high autoantibody titers to oxidized low density lipoprotein, showed a striking time-dependent increase in binding to CL that was exposed to air for increasing periods of time. Monoclonal antibodies to oxidized LDL, cloned from the apo E-deficient mice, also bound to oxidized CL. Both sera and affinity-purified aCL-IgG from APS patients bound to CL progressively as it was oxidized. However, the monoclonal antibodies from apo E-deficient mice, or sera or aCL-IgG from APS patients did not bind to a reduced CL analog that was unable to undergo peroxidation. These data demonstrate that many aPL are directed at neoepitopes of oxidized phospholipids, and suggest that oxidative events may be important in the pathophysiology of APS. In turn, this suggests new therapeutic strategies, possibly including intensive antioxidant therapy.

Plasma cell dyscrasia with polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes: the POEMS syndrome. Report on two cases and a review of the literature.

Two patients with plasma cell dyscrasias, manifested by osteosclerotic bone lesions and small amounts of M protein, and a complicating multi-system disorder are described. Their features of severe sensory-motor polyneuropathy, organomegaly, endocrine dysfunction, anasarca, elevated CSF protein, and skin hyperpigmentation are similar to a clinical syndrome reported primarily in Japanese men. Two previously unrecognized findings--hyperprolactinemia and an unusual radiographic abnormality of fluffy, spiculated bony proliferation--may facilitate recognition of the syndrome. The relationship of these various manifestations to the plasma cell dyscrasia is unknown, but a number of possibilities are discussed.

Generation of human cytomegalovirus-specific cytotoxic T-lymphocytes in a short-term culture.

A method to generate human cytomegalovirus (HCMV)-specific CTL (cytotoxic T lymphocytes) from human peripheral blood mononuclear cells is described. This assay is unique in comparison with other methods reported to date, because it only requires a short-term (6 days) coculture of PBM and autologous infected fibroblasts without the addition of exogenous IL-2 (interleukin-2) and nevertheless is sensitive enough to determine HCMV-specific killing in a short (6 h) 51Cr-release assay using autologous HCMV-infected fibroblasts as targets. The virus-specific killing is mediated by CTL of the CD8 phenotype and it can be inhibited by a HLA class I monoclonal antibody. The sensitivity of the assay can be significantly enhanced by pretreating the targets with interferon-gamma (IFN-gamma) prior to infection with HCMV. HCMV-specific 51Cr-release is more than doubled when the IFN-gamma pretreated targets are used. This increase is mostly due to enhanced sensitivity of the fibroblasts to killing mediated by CD8-positive CTL, but some killing can be attributed to CTL of the CD4 phenotype.