VAV1 regulates experimental autoimmune arthritis and is associated with anti-CCP negative rheumatoid arthritis.

Rheumatoid arthritis (RA) patients can be stratified into two subgroups defined by the presence or absence of antibodies against citrullinated circular peptides (anti-CCP) with most of the genetic association found in anti-CCP positive RA. Here we addressed the role of VAV1, previously associated to multiple sclerosis (MS), in the pathogenesis of RA in experimental models and in a genetic association study. Experimental arthritis triggered by pristane or collagen type II was induced in DA rats and in the DA.BN-R25 congenic line that carries a polymorphism in Vav1. Difference in arthritis severity was observed only after immunization with pristane. In a case-control study, 34 SNPs from VAV1 locus were analyzed by Immunochip genotyping in 11475 RA patients (7573 anti-CCP positive and 3902 negative) and 15,870 controls in six cohorts of European Caucasians. A combination of the previous MS-associated haplotype and two additional SNPs was associated with anti-CCP negative RA (alleles G-G-A-A of rs682626-rs2546133-rs2617822-rs12979659, OR=1.13, P=1.27 × 10-5). The same markers also contributed to activity of RA at baseline with the strongest association in the anti-CCP negative group for the rs682626-rs12979659 G-A haplotype (ß=-0.283, P=0.0048). Our study suggests a role for VAV1 and T-cell signaling in the pathology of anti-CCP-negative RA.

Genetic control of HgCl2-induced IgE and autoimmunity by a 117-kb interval on rat chromosome 9 through CD4 CD45RChigh T cells.

Gold or mercury salts trigger a dramatic IgE response and a CD4 T-cell-dependent nephropathy in Brown-Norway (BN), but not in Lewis (LEW) rats. We previously identified the 1.1-Mb Iresp3 (immunoglobin response QTL3) locus on chromosome 9 that controls these gold salt-triggered immune disorders. In the present work, we investigated the genetic control of HgCl(2)-induced immunological disorders and assessed the relative contribution of the CD45RC(high) and CD45RC(low) CD4 T-cell subpopulations in this control. By using interval-specific congenic lines, we narrowed down Iresp3 locus to 117-kb and showed that BN rats congenic for the LEW 117-kb were protected from HgCl(2)-triggered IgE response and nephropathy. This 117-kb interval also controls CD45RC expression by CD4 T cells and the ability of CD45RC(high) CD4 T cells to trigger the autoimmune disorders resulting from HgCl(2) administration. This 117-kb region contains four genes, including Vav1, a strong candidate gene according to its cellular function and exclusive expression in hematopoietic cells. Thus, this study highlights the role of the CD45RC(high) CD4 T-cell subpopulation in the opposite susceptibility of BN and LEW rats to HgCl(2)-triggered immune disorders and identifies a 117-kb interval on chromosome 9 that has a key role in their functions.

Release of DNA in circulating blood and induction of anti-DNA antibodies after injection of bacterial lipopolysaccharides.

The present data demonstrate the induction of antisingle-stranded (SS) DNA and antidouble-stranded DNA antibodies in various strains of mice, including athymic C57BL/6 nude mice, after the injection of bacterial lipopolysaccharide (LPS). This anti-DNA response is dose dependent and varies quantitatively according to the strain of the injected mice. It is not correlated to the H-2 histocompatibility locus nor to the immune response to LPS. The lipid A fraction appears to be the active part of the LPS molecule for this particular effect. In addition, it was found that DNA is released in circulating blood a few hours after the injection of LPS. Most of the DNA released has physicochemical and immunochemical characteristics of SS DNA. Therefore, the anti-DNA response induced by injections of LPS may be the result of a release of DNA in a particularly immunogenic form at a time when the immune system, in particular the B lymphocytes, is rendered capable by LPS of developing an immune response to such a soluble antigen. These effects of LPS may account for the triggering or the exacerbation of ante-DNA antibodies during infections with gram-negative bacteria, and a similar mechanism may be involved in the pathogenesis of systemic lupus erythematosus.

Features of systemic lupus erythematosus in mice injected with bacterial lipopolysaccharides: identificantion of circulating DNA and renal localization of DNA-anti-DNA complexes.

After injection of lipopolysaccharides (LPS) in mice, there is first a release of DNA into plasma and secondly an induction of anti-DNA antibodies. The circulating DNA was purified from plasma and physico-immunochemically characterized. This DNA has a similar density to mammalian cellular DNA,is 4--6S insize, and probably represents a mixture of single-stranded DNA (SSDNA) and double-stranded DNA (DSDNA) or DSDNA with some single-stranded regions. This purified DNA was shown to react with anti-DNA antibodies which appeared as early as 3 days after a single injection of LPS in mice. In serum, DNA-anti-DNA complexes were not detected, although unidentified circulating immune complex-like material was demonstrated 5-8 days after the injection of LPS. In tissues, particularly in renal glomeruli, fine granular immune complex-type immunoglobulin deposits appeared along the glomerular capillary walls and in the mesangium 3 days after the injection of LPS. There is a direct correlation between the level of anti-DNA antibodies and the intensity of glomerular deposits and about 40% of immunoglobulins eluted from kidneys are anti-DNA antibodies, indicating that some of the immune complexes localized in kidneys are DNA-anti-DNA complexes. Based on these observations, the following hypothetical mechanism for the glomerular localization of DNA-anti-DNA complexes after the injection of LPS in mice is proposed. First, DNA, which has been released in circulating blood after injection of LPS, might bind to renal glomeruli, probably on glomerular basement membranes (GBM) through a high affinity of GBM for DNA; secondly, circulating anti-DNA antibodies, which appear later, might react with the glomerular-bound DNA and form immune complexes independently of circulating immune complexes. However, the possibility of direct deposition of immune complexes is not ruled out.

Clinical relevance of circulating immune complexes in human leukemia. Association in acute leukemia of the presence of immune complexes with unfavorable prognosis.

The occurrence of circulating immune complexes was investigated in 467 serum samples from 230 leukemia patients using the [(125)I]Clq-binding test. There was an increased serum [(125)I]Clq-binding activity in 40% of patients with acute myeloid leukemia, 23% with acute lymphatic leukemia, 46% in blastic crisis of chronic myeloid leukemia, 12% with chronic lymphatic leukemia, and 13% with chronic myeloid leukemia. In 48 patients, serum was also tested for soluble immune complexes by the Raji cell radioassay; the correlation between results of the two tests was significant. The Clq-binding material had properties identical with those of immune complexes. It sedimented as 14-28s material on sucrose density gradient. It contained IgG which could be dissociated at acid pH. Its Clq-binding properties could be removed after passage through anti-IgG immuno-absorbant or after a mild reduction-alkylation treatment, but were not sensitive to deoxyribonuclease treatment. Circulating immune complexes were found most commonly during the blastic stage of leukemia.Remission took place in 75.4% of patients with no detectable circulating immune complexes at the onset of acute leukemia, but in only 32.7% of those with detected complexes during this period. Median survival times of the former group of patients were more than 18 mo in acute myeloid leukemia and acute lymphatic leukemia and more than 8(1/2) mo in blastic crisis of chronic myeloid leukemia. The corresponding median survival times in the latter patient group were 64, 135, and 90 days. These findings were unrelated to prognostic features already known.

LDL and acetyl-LDL inhibit the NK activity and are taken up by CD56+ lymphocytes.

The effect of LDL and modified LDL (acetyl-LDL) was studied on human natural killer cell-mediated cytotoxicity against K562 cells. Incubation for 24 h of peripheral blood lymphocytes (PBL) with a high concentration (200 micrograms/ml) of LDL decreased the NK activity in some donors. After acetylation of the LDL protein (apoB), the modified-LDL systematically inhibited the NK function of PBL in a time- and dose-dependent manner. Inhibition mediated by acetyl-LDL (AcLDL) was significantly greater than that of LDL, indicating that the apoB modification can mediate the inhibition of the NK function. AcLDL also inhibited the NK activity of peripheral blood mononuclear cells, suggesting that, under our experimental conditions, monocytes are not efficient enough to protect NK cells against the adverse effects of modified-LDL. With a cytofluorimetric analysis, the internalization of acetyl-LDL by PBL was demonstrated and was only 3-4 times lower than LDL internalization in lymphocytes. It appeared to be time, temperature and dose dependent, saturable and different from the internalization mediated by the known scavenger receptors. Finally, CD14- CD3+ lymphocytes and CD14- CD56+ lymphocytes were able to internalize AcLDL in the same way. Our results suggest that in some in vivo circumstances, when the LDL concentration and/or the modified-LDL/LDL ratio increase in tissues, lipoproteins are internalized by NK cells and also can induce adverse effects on the NK function.

Study of LDL and acetylated LDL endocytosis by mononuclear cells in HIV infection.

Activated lymphocytes have a high level of low density lipoprotein (LDL) uptake as compared to resting lymphocytes, whereas scavenger receptors for acetylated LDL (Ac-LDL) are expressed on limited number of immune cells, i.e., monocytes/macrophages. The endocytosis of LDL and Ac-LDL by mononuclear cells was studied during in vitro and in vivo HIV infection, in order to use LDL and Ac-LDL as carriers of antiviral and/or immunomodulatory drugs towards lymphocytes and monocytes. The uptake of LDL and Ac-LDL was analyzed by cytofluorimetry. LDL endocytosis in PHA/IL2-activated lymphocytes was higher than in resting lymphocytes. In vitro HIV infection of PHA/IL2-activated lymphocytes did not alter the high LDL endocytosis in lymphocytes. CD4+ and CD8+ cells. In a group of 12 symptomatic patients there was no alteration of LDL endocytosis in lymphocytes, CD4 and CD8 lymphocytes. In another group of 23 individuals, the Ac-LDL endocytosis mediated by CD14+ monocytes was unaltered in asymptomatic patients (n = 6) and in some symptomatic patients (n = 6, CD14+ cells > 100/mm3). On the contrary, in other symptomatic patients (n = 11, CD14+ cells < 100/mm3), the number of Ac-LDL+ CD14+ cells decreased, whereas their efficiency of Ac-LDL endocytosis increased as compared to those of other HIV+ patients. In conclusion, the use of lipoproteins as carriers to increase the drug delivery to CD4+ lymphocytes and to CD14+ monocytes can be envisaged, since: (i) the LDL endocytosis was not impaired in CD4 lymphocytes of HIV+ patients, and (ii) the Ac-LDL uptake by monocytes was altered only in some patients of stage IV.

Plasma DNA as a marker of cancerous cell death. Investigations in patients suffering from lung cancer and in nude mice bearing human tumours.

Plasma DNA that circulates mainly as mononucleosomes is a cell death marker. Its significance and prognostic value in cancer as compared to other tumour markers was investigated in 68 patients hospitalised for lung cancers. Prognostic values of the various studied parameters were evaluated using the Cox's model. The cellular origin of plasma DNA was further investigated in nude mice transplanted with human lung adenocarcinoma. Plasma DNA concentrations were increased in cancer patients as compared to normal subjects (P < 0.01). They were higher in patients with extended (Stage 4) disease than in patients with limited stage disease (P < 0.05). Plasma DNA concentrations, serum lactate dehydrogenase activities and neuron-specific enolase concentrations were correlated all together in small cell lung carcinoma (SCLC) and in non-SCLC. Similar relationships were found between survival and each of these three cell death/tumour markers (P < 0.02-0.005). Plasma DNA from mice bearing human tumour hybridised with both mouse and human plasma DNA, while plasma DNA from endotoxin-injected mice hybridised only with mouse plasma DNA. In conclusion, in patients suffering from lung cancer, plasma DNA as well as LDH and NSE represent cell death markers that are correlated with survival. At a time when apoptosis pathways appear to be potential targets for cancer therapy, plasma DNA is a cell death/tumour marker that should be taken into account in studying the cancerous process in human diseases.

Quantitation of blood plasma DNA as an index of in vivo cytotoxicity.

Cell death triggers the release into extracellular spaces of products of chromatin catabolism, particularly of DNA. A sensitive DNA assay has been used to investigate in the mouse whether the quantitation of plasma DNA may be used as an index of in vivo cytotoxicity. It has been found that toxic doses of bacterial lipopolysaccharide, HgCl2, CCl4, cyclophosphamide and hydroxyurea, are responsible for the release of extracellular DNA in plasma, in a dose dependent relationship. In conclusion, quantitation of extracellular DNA may be used for investigating in vivo cell death phenomena induced by toxic agents and drugs. Such a method could be applied to toxicological studies in animals and man.

Urinary DNA as an indicator of nephrotoxicity caused by endotoxin and gentamicin in mice.

Extracellular DNA is a non-specific marker of cell death. Urinary DNA, as an indicator of nephrotoxicity, was investigated in endotoxin/gentamicin-injected mice. In mice injected both with endotoxin (15 mg/kg) and gentamicin (80 mg/kg), urinary DNA concentration was markedly increased for several days; in contrast, there was at most a slight and transient excretion of DNA in mice receiving gentamicin or endotoxin alone. Plasma DNA concentrations increased for 24-48 h in endotoxin-injected mice, then decreased rapidly. Mice injected with gentamicin and endotoxin showed widespread and severe kidney lesions with tubular cell necrosis and intraluminal casts while mice receiving gentamicin or endotoxin alone showed at most few and mild lesions. In mice receiving lower doses of endotoxin (5-10 mg/kg) and 80 mg/kg gentamicin, urinary DNA peaked at 72-96 h, at a time when plasma DNA had returned to normal concentrations. Maximal urinary DNA concentrations depended upon endotoxin dose. In conclusion, urinary DNA is a marker of definite cell death occurring in the urinary tract and could represent a new indicator of nephrotoxicity in clinical and experimental situations.

Detection of nucleosome-IgG immune complexes in ascites from mice transplanted with anti-DNA antibody-secreting hybridomas and in plasma from MRL-lpr/lpr mice.

Autoantibodies directed against chromatin components characterize lupus diseases. Immune complexes made of these autoantibodies bound to nucleosomes released from dead cells could play some pathogenic role. The aims of this study were to investigate if nucleosome-IgG complexes could contaminate IgG anti-DNA MoAb preparations, and if such complexes circulate in lupus diseases. A new method was set up using preformed nucleosome-IgG complexes. Complexes were adsorbed onto microplate through Fc binding and nucleosomal DNA was detected by internal incorporation of labelled nucleotide. Using this method, high amounts of complexes were found in ascites from mice transplanted with anti-DNA antibody-secreting hybridomas. In some ascites, nucleosome was found to be strongly associated with the MoAb, confirming that nucleosome-IgG complexes could contaminate monoclonal autoantibody preparations. In MRL-lpr/lpr mice, nucleosome-IgG complexes were detected at 16-24 weeks of age at a time when kidney lesions are rapidly worsening, raising the question of their pathogenic significance.

Beta blockers may induce in C57BL/6 mice a polyclonal activation of lymphocytes which is not mediated by beta adrenergic receptors.

The in vivo immune effects of nine beta (beta)-blockers are studied in C5B1/6 mice after 7 days of treatment. Humoral and cellular studies indicate that several beta-blocking agents, particularly pindolol and acebutolol, may induce a polyclonal activation of lymphocytes. It may involve all the classes and subclasses of immunoglobulins with a preferential effect on IgA and IgG2a. Further investigations, conducted using the laevorotatory and dextrorotatory forms of pindolol indicate that this immune effect is not mediated by specific beta-adrenergic receptors.

Quantification of large C3 fragments as an index of complement activation in mice. II. In vivo studies.

A method which enables to evaluate the mouse C3 activation has been used to study the activation of the complement system in C57Bl/6 mice injected with cobra venom factor, with Escherichia coli, and with bacterial lipopolysaccharide. It was found that this method was able to detect the complement activation early, at a time when total levels of C3-reacting molecules had not yet decreased.

Polyclonal stimulation of lymphocytes induced by beta blockers in the mouse: influences of age, sex and strain.

The immune effects of acebutolol, a beta-blocker, are investigated in various strains of mice. In 6-12 weeks old C57Bl/6 female mice, an increase in the number of immunoglobulin secreting cells is observed only at the age of 9 weeks. At that age, such an effect is seen neither in C57Bl/6 male mice nor in female mice from 6 other strains including OF1 outbred mice. An increase in anti-trinitrophenol plaque forming cells is found in 9 week old C57Bl/6, Balb/c and CBA female mice.

Quantitative clonal analysis of the B cell repertoire in human lupus.

To gain further insight into the origin of autoantibody hyperproduction in human lupus, we quantitated the B cell repertoire toward exogenous and self-antigens. Using the Spot-ELISA method and two panels of nine exogenous and 10 self-antigens, we found that the normal human immune repertoire comprises a high frequency of B cell precursors secreting IgM antibodies to self- and exogenous determinants. This repertoire was markedly deficient in precursors producing IgG able to bind self-antigens. In lupus patients, the absolute numbers of clone precursors of the immune repertoire expressing IgM receptors whose paratopes impart affinity to self- and exogenous determinants were higher than in control individuals. Additionally, IgG antibody-forming cell precursors with binding specificity for lupus-associated antigens were detectable in the repertoire of these patients. Based on these results, we propose that hyperproduction of human lupus-associated autoantibodies arises in a two-stage mechanism whereby a general activation of the multireactive immune B cell repertoire precedes an oligospecific expansion of selected B cell clonotypes.

Dihydroergotamine: an effective treatment for postural hypotension due to antihypertensive drugs (ganglion-blocking agents excepted).

In this study the effect of DHE on postural hypotension induced by major antihypertensive drugs was evaluated in 40 patients. 30 patients were treated with methyldopa, five with guanoxan sulphate and five with bethanidine sulphate. To obtain a more accurate picture of the effectiveness of DHE and to test the reproducibility of its effect, each patient was observed during five separate, successive periods: in the first period the antihypertensive agent was given alone; in the second period it was given along with placebo; in the third period it was given with DHE; in the fourth period the antihypertensive agent was given alone again; and in the fifth period it was again given with DHE. In the third and in the fifth period, DHE was administered at the same time as the antihypertensive agent in a dose of 9-15 mg/24 h (3-5 mg three times daily). The first dose was given 1 h before rising, and the daily dosage was progressively increased. The beneficial effect of DHE on postural hypotension was evaluated by assessing the clinical symptoms in a semiquantitative manner and by measuring the arterial blood pressure and heart rate in a recumbent and standing position. The results were classified as follows: excellent, good, moderate and no response. In most cases, DHE was found to be an effective drug for the treatment of postural hypotension, an improvement in clinical symptoms being noted in 57.5% of patients tested (excellent and good results). In these patients the standing arterial blood pressure showed a significant response (p less than 0.01). DHE did not interfere with the therapeutic effect of the antihypertensive agents. Furthermore, DHE did not affect the heart rate, nor did it give rise to any adverse reactions.

Features of the immune response to DNA in mice. I. Genetic control.

The genetic control of the immune response to DNA was studied in various strains of mice F1 hybrids and corresponding back-crosses immunized with single stranded DNA complexed to methylated bovine serum albumin. Anti-DNA antibody response was measured by radioimmuno-logical technique. High responder, low responder, and intermediate responder strains were found and the ability to respond to DNA was characterized as a dominant genetic trait which is not linked to the major locus of histocompatibility. Studies in back-crosses suggested that this immune response is under multigenic control. High responder mice produce both anti-double stranded DNA and anti-single stranded DNA 7S and 19S antibodies, while low responder mice produce mainly anti-single stranded DNA 19S antibodies.

Nucleosome-dependent escape of tumor cells from natural-killer-mediated lysis: nucleosomes are taken up by target cells and act at a postconjugation level.

Our previous data suggested that chromatin fragments released from dead cells into the extracellular medium could be involved in the impairment of natural-killer (NK)-mediated cytotoxicity reported in cancer patients. In the present study, an inhibition of the NK-mediated lysis was obtained in vitro by nucleosome addition to different tumor target cells, independently of their sensitivity to NK-mediated lysis. We observed a rapid endocytosis and degradation of nucleosomes by K562 tumor target cells and (although to a much lesser extent) a binding to a subpopulation of lymphocytes. Nucleosomes impaired neither the conjugation step nor the expression of adhesion molecules at the effector (CD11a, CD18, CD2) or target (CD54, CD58) cell surface. On the contrary, flow-cytometry analysis of the conjugation suggested that nucleosomes might stabilize the conjugates. Investigations of the killing process showed that nucleosomes decreased the NK cytotoxic potential without modifying Ca2+-dependent lethal-hit-delivery kinetics. The cytotoxic potential was not restored by increasing the available magnesium and calcium concentrations in the extracellular medium. Taken together, the results suggest that the inhibition of NK-mediated lysis by nucleosomes may result from alterations of the NK mechanism at the postconjugation level and after lethal-hit delivery. Hence, the inhibition could involve a delay in the recycling of effector cells, or a resistance of tumor target cells to NK cells.