Association of EBF1, FAM167A(C8orf13)-BLK and TNFSF4 gene variants with primary Sjögren's syndrome.

We performed a candidate gene association study in 540 patients with primary Sjögren's Syndrome (SS) from Sweden (n=344) and Norway (n=196) and 532 controls (n=319 Swedish, n=213 Norwegian). A total of 1139 single-nucleotide polymorphisms (SNPs) in 84 genes were analyzed. In the meta-analysis of the Swedish and Norwegian cohorts, we found high signals for association between primary SS and SNPs in three gene loci, not previously associated with primary SS. These are the early B-cell factor 1 (EBF1) gene, P=9.9 × 10(-5), OR 1.68, the family with sequence similarity 167 member A-B-lymphoid tyrosine kinase (FAM167A-BLK) locus, P=4.7 × 10(-4), OR 1.37 and the tumor necrosis factor superfamily (TNFSF4=Ox40L) gene, P=7.4 × 10(-4), OR 1.34. We also confirmed the association between primary SS and the IRF5/TNPO3 locus and the STAT4 gene. We found no association between the SNPs in these five genes and the presence of anti-SSA/anti-SSB antibodies. EBF1, BLK and TNFSF4 are all involved in B-cell differentiation and activation, and we conclude that polymorphisms in several susceptibility genes in the immune system contribute to the pathogenesis of primary SS.

Antibody and immunoglobulin production at the cellular level in bursectomized-irradiated chickens.

The effect of bursectomy combined with sublethal X-irradiation in the newly hatched chicken on the immunoglobulin and antibody producing capacity in later life was investigated. The previous findings of a significant incidence of hypogammaglobulinemia in such animals were confirmed. Spleen cells from severely hypogammaglobulinemic animals synthesized and secreted little or no immunoglobulin. Such spleen lymphoid cells contained fewer immunoglobulin antigenic determinants than spleen cells from irradiated control animals as evidenced by their relative inability to respond by an increased DNA synthesis after in vitro culture with rabbit antiserum to chicken immunoglobulin. Therefore, the deficiency in the immunoglobulin synthesis extends not only to actively secreting cells such as plasma cells, but to the entire lymphoid cell population. As expected, most irradiated-bursectomized chickens, irrespective of plasma immunoglobulin levels failed to produce detectable amount of circulating antibodies to Brucella abortus antigen in the primary immune response. Severely hypogammaglobulinemic animals were completely unable to elaborate any plaque forming cells (PFC) in the primary response to sheep red blood cells (SRBC). The results of this investigation support the contention that in the severely hypogammaglobulinemic bursectomized-irradiated chicken the entire antibody producing and immunoglobulin producing cell line is absent. The possibility remains that precursor or stem cells are present but are not appropriately directed to antibody synthesis by other cell types.

Additive effects of the major risk alleles of IRF5 and STAT4 in primary Sjögren's syndrome.

Primary Sjögren's syndrome (SS) shares many features with systemic lupus erythematosus (SLE). Here we investigated the association of the three major polymorphisms in IRF5 and STAT4 found to be associated with SLE, in patients from Sweden and Norway with primary SS. These polymorphisms are a 5-bp CGGGG indel in the promoter of IRF5, the single nucleotide polymorphism (SNP) rs10488631 downstream of IRF5 and the STAT4 SNP rs7582694, which tags the major risk haplotype of STAT4. We observed strong signals for association between all three polymorphisms and primary SS, with odds ratios (ORs) >1.4 and P-values <0.01. We also found a strong additive effect of the three risk alleles of IRF5 and STAT4 with an overall significance between the number of risk alleles and primary SS of P=2.5 x 10(-9). The OR for primary SS increased in an additive manner, with an average increase in OR of 1.78. For carriers of two risk alleles, the OR for primary SS is 1.43, whereas carriers of five risk alleles have an OR of 6.78. IRF5 and STAT4 are components of the type I IFN system, and our findings emphasize the importance of this system in the etiopathogenesis of primary SS.

Interferon-beta is required for interferon-alpha production in mouse fibroblasts.

The type I interferons--interferon-alpha (IFN-alpha) and interferon-beta (IFN-beta)--are critical for protection against viruses during the acute stage of viral infection [1,2]. Furthermore, type I interferons have been implicated as important mediators in the regulation of lymphocyte development [3], immune responses [4,5] and the maintenance of immunological memory of cytotoxic T cells [6,7]. The different IFN-alpha subtypes are encoded by 12 genes in the mouse [8] whereas IFN-beta is encoded by only one gene [9]. IFN-alpha and IFN-beta have a high degree of sequence homology and are thought to interact with the same surface receptor on target cells [10,11]. As an approach to analysing the different biological functions of IFN-alpha and IFN-beta, we have generated a mouse strain with an inactivated IFN-beta gene. We report here that embryonic fibroblasts from such mice produce neither IFN-beta nor IFN-alpha upon Sendal virus infection, whereas the production of IFN-alpha by leukocytes from the same strain of mice is intact. IFN-alpha production in embryonic fibroblasts from IFN-beta-/- mice could be rescued by 'priming' the cells using exogenous IFN-beta. These results imply a unique role for IFN-beta in the induction of type I interferons in peripheral tissues.

Repression of beta interferon gene expression in virus-infected cells is correlated with a poly(A) tail elongation.

Expression of beta interferon (IFN-beta) is transiently induced when Namalwa B cells (Burkitt lymphoma cell line) are infected by Sendai virus. In this study, we found that an elongation of the IFN-beta mRNA could be detected in virus-infected cells and that such a modification was not observed when the IFN-beta transcript was induced by a nonviral agent, poly(I-C). Treatment of the cells with a transcriptional inhibitor (actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) resulted in further elongation of the transcript. Characterization of the elongated IFN-beta transcript by primer extension and RNase H treatment showed that the modification was a result of an elongated poly(A) tail of up to 400 nucleotides. We conclude that the poly(A) tail elongation of the IFN-beta transcript is associated with the viral infection. Furthermore, the presence of the elongated IFN-beta transcript correlated with a decrease of IFN-beta protein in the medium and in cell extracts. Sucrose gradient analysis of cytoplasmic extracts showed that IFN-beta transcripts with elongated poly(A) tails were found in the nonpolysomal fractions, whereas the shorter transcripts could be detected in both polysomal and nonpolysomal fractions. A longer form of the IFN-beta mRNA was also found in the nonpolysomal fractions of cells not treated with transcriptional inhibitors. Thus, the observed regulation of IFN-beta mRNA is not entirely dependent on the inhibition of transcription. To our knowledge, this study provides the first example of a poly(A) tail elongation in somatic cells that negatively influences gene expression in vivo.

Natural cell-mediated immunity to lymphoma cells. I. Characteristics of effector cells in a cytostasis assay in vitro.

Spleen cells from normal, nonimmune, CBA or (CBA X AKR)F1 mice markedly and rapidly inhibited the incorporation of [3H]thymidine by two different T-cell lymphomas in an in vitro cytostasis assay. These were the I-529 lymphoma of spontaneous AKR origin and the Moloney murine leukemia virus-induced YAC lymphoma of A mouse origin. Spleen cells were the most efficient inhibitors for both types of target cells, whereas lymph node cells were much less active and thymus cells showed little or no activity. Granulocytes, as well as conventional T- and B-lymphocytes, were excluded as important contributors to the cytostatic cell population. Spleen cells were separated on nylon wool, Sephadex G-10 columns, or plastic petri dishes and tested for activity in the cytostasis assay or for cytotoxicity against 51Cr-labeled lymphoma target cells. Adherent cells carried almost all cytostatic activity against the AKR lymphoma but also showed significant cytotoxic activity against these target cells. In addition, the cytostatic activity against the YAC lymphoma was mainly due to adherent spleen cells, but nonadherent cells were relatively more active against this target than against I-529 cells. Such nonadherent spleen cells further showed increased cytotoxic activity, compared to the whole spleen cell population.

Treatment of malignant carcinoid tumors with recombinant interferon alfa-2b: development of neutralizing interferon antibodies and possible loss of antitumor activity.

Twenty patients with malignant carcinoid tumors were treated for 6 months with recombinant interferon alfa-2b (IFN alpha-2b; Intron-A; Schering Corp., Bloomfield, NJ) at a mean dose of 5.9 megaunits three times per week. Eleven of the 20 patients (55%) had a greater than 50% reduction of tumor markers (urinary 5-hydroxyindoleacetic acid or plasma neuropeptide K), showing objective tumor response. Six patients (30%) had stable disease with no significant change in tumor markers or tumor size, and three (15%) had progressive disease with an increase in tumor markers and size. These results are similar to those reported earlier for treatment with natural leukocyte IFN in patients with carcinoid tumors. Only two patients (35%) had a slight reduction of tumor size after 6 months of treatment. Three patients developed neutralizing antibodies to IFN alpha-2b. Two of these patients initially showed an objective response, which lasted until IFN antibodies developed. In one of these patients, a change to human leukocyte IFN resulted in normalization of antibody titers within 3 months, and the patient had a second objective clinical response. There was no correlation between development of IFN antibodies and development of autoimmune phenomena such as increased titers of antinuclear antibodies or thyroid autoantibodies. IFN alpha-2b seems to be as potent as human leukocyte IFN in the treatment of patients with malignant carcinoid tumors, but it is important to recognize that antibodies neutralizing IFN may develop in some patients, with concomitant loss of antitumor effects. A change to natural leukocyte IFN might be beneficial in these patients.

Multiple sclerosis: interferon-beta induces CD123(+)BDCA2- dendritic cells that produce IL-6 and IL-10 and have no enhanced type I interferon production.

Interferon-beta (IFN-beta), an approved drug for multiple sclerosis (MS), acts on dendritic cells (DC) by suppressing IL-12p40 and increasing IL-10. This results in Th2-biased immune responses. The nature of IFN-beta-modulated DC remains elusive. Previously, we observed that IFN-beta dose dependently induces expression of CD123, i.e., a classical marker for plasmacytoid DC, on human blood monocyte-derived myeloid DC. Such IFN-beta-modulated DCs produce predominantly IL-10 but are IL-12 deficient, with potent Th2 promotion. In the present study, we further characterize IFN-beta-modulated DC by using recently identified blood DC antigens (BDCA), and investigate their ability to produce type I IFN in response to virus stimulation. We show that IFN-beta induces development of CD123+ DC from human blood monocytes, which coexpress BDCA4+ but are negative for BDCA2-, a specific marker for plasmacytoid DC. Such IFN-beta-modulated DC can produce IL-6 and IL-10 but not IL-12p40, and have no enhanced IFN-alpha and IFN-beta production. The findings indicate that IFN-beta-modulated DCs represent a myeloid DC subset with diminished CD11c, BDCA-1 and CD1a expression. They may promote Th2 and B cell differentiation through IL-6 and IL-10 production, and suppression of IL-12p40, but they have no enhanced antiviral capacity.

Stimulation of natural interferon-alpha/beta-producing cells by Staphylococcus aureus.

Human peripheral blood mononuclear cells (PBMCs) produced high levels of antiviral activity, as determined by bioassay, when stimulated by Staphylococcus aureus Cowan I (SAC) and E. coli. Specific immunoassays demonstrated the presence of both IFN-alpha and gamma and, for SAC, also low levels of IFN-beta. The frequencies of SAC-induced IF N-alpha-producing cells (IPCs) were up to 1-2 per 10(3) PBMCs. These IPCs expressed the HLA-DR and CD4 antigens but not CD3, CD14, or CD19, thus resembling the natural IFN-alpha-producing cells (NIPC). The SAC was more efficient as IFN inducer when heat killed than when streptomycin inhibited. The SAC was inhibitory to virally induced IFN-alpha responses, in particular when streptomycin inhibited. Both pronase treatment and mechanical disruption of SAC cells abolished their capacity to induce IFN-alpha production. Staphylococcal strains lacking or expressing low levels of protein A (SpA) showed a decreased ability to induce IFN-alpha production. However, purified SpA did not itself induce IFN-alpha. Possibly, SpA together with other bacterial surface proteins is important for the capacity of SAC to induce IFN-alpha production in NIPC.

Development of a highly purified multicomponent leukocyte IFN-alpha product.

A purification process was developed to obtain a human interferon- alpha (IFN-alpha) product that contains all major IFN-alpha subtypes produced by human leukocytes. The purification was accomplished by immunoaffinity chromatography using two monoclonal antibodies (mAb) and gel filtration. The process comprised two effective virus inactivation steps, solvent detergent treatment, and incubation at low pH, and the purified product was filtered with a 15-nm pore size virus removal filter. The overall yield of IFN-alpha in the process was about 60% when starting from the culture supernatant of Sendai virus-induced human leukocytes. The specific activity was about 1.0 x 10(8) IU/mg. The level of DNA and protein impurities including mouse IgG was very low. The product contained seven main subtypes: IFN-alpha 1, IFN-alpha 2, IFN-alpha 8, IFN-alpha 10, IFN-alpha 14, IFN-alpha 17, and IFN-alpha 21. The subtypes IFN-alpha 4 and IFN-alpha 7 were minor components. Reverse-phase HPLC indicated a constant subtype composition for the product from batch to batch. Stabilization of the pure IFN-alpha solution with albumin and Tween 80 was compared. In virus filtration, a better yield and higher filtration capacity were obtained with Tween. The addition of albumin resulted in the formation of IFN-albumin aggregates. During long-term storage, IFN-alpha was stable in both solutions for 2 years at 2-8 degrees C. The new method makes it possible to extensively purify all major IFN-alpha subtypes and obtain a virus-safe and stable product with a constant subtype composition.

Herpes simplex virus induces appearance of interferon-alpha/beta-producing cells and partially interferon-alpha/beta-dependent accumulation of leukocytes in murine regional lymph nodes.

As in vivo experimental system involving the local induction of interferon-alpha/beta (IFN-alpha/beta) responses was established in mice by injecting s.c. ultraviolet (UV)-inactivated herpes simplex virus (HSV) in the right ears, the left ears receiving phosphate-buffered saline (PBS) as a control. Circulating IFN-alpha/beta was present in blood as early as 6 h postinjection, and little or none was found 24 h postinjection. Identification of IFN-alpha/beta-producing cells, carried out by immunohistochemistry and in situ hybridization, demonstrated that the IFN response occurred mainly in the lymph node draining the HSV-injected ear and not in the contralateral lymph node. Occasionally, IFN-alpha/beta-producing cells were found in the spleen and in the skin. The injected HSV caused an inflammatory reaction in the skin and an almost threefold enlargement of the draining lymph node within 6 h. The latter was characterized by a general accumulation of all major lymphocyte subsets and a striking infiltration of neutrophils. Injection s.c. of neutralizing anti-IFN-alpha/beta antibodies before HSV injection reduced the increase in size of the draining lymph node by approximately 50% at 6 h, and no significant effects were seen at 24 h. The localization of cells producing IFN-alpha/beta in the lymph node and the capacity of such IFN-alpha/beta to at least partially mediate an early accumulation of cells suggest that the local IFN-alpha/beta response may have an important role in the initiation of early antiviral immune responses.

Filter spot-ELISA for the enumeration of interferon-alpha antibody-secreting cells.

A filter spot-ELISA was developed for the enumeration of interferon-alpha (IFN-alpha) antibody secretion by murine and human cells. Various human IFN-alpha subtypes were coated onto nitrocellulose membranes by means of broad specificity bovine antibodies. Membranes were blocked with milk proteins, and antibodies released by individual cells during a 3 h culture period were visualized as distinct spots using peroxidase-conjugated, species-specific anti-immunoglobulin antibodies and diaminobenzidine/hydrogen peroxide substrate solution. The spot-ELISA is rapid, easy to perform, and highly sensitive. Possible applications include frequency estimates of IFN-alpha antibody-secreting cells in blood, tissues and hybridoma cultures as well as the evaluation of specificity and immunoglobulin class of the respective antibodies.

Monoclonal antibody two-site ELISA for human IFN-gamma. Adaptation for determinations in human serum or plasma.

Mouse monoclonal antibodies (mAbs) against human interferon-gamma (IFN-gamma) were produced after immunization with recombinant IFN-gamma. Two mAbs (1-D1K and 7-B6-1) recognizing distinct epitopes on natural IFN-gamma were selected for the development of a two-site ELISA. The sensitivity was similar for IFN-gamma diluted in PBS with 1% bovine albumin, spent culture medium or fetal calf serum but reduced to approximately 50% when diluted in normal human serum. Individual normal human sera were tested and three of 14 gave false reactivities in the ELISA. One serum factor with major impact on the individual variation and the decreased sensitivity could be adsorbed to and eluted from protein A-Sepharose. Based on these observations we established a new ELISA protocol which made it possible to test for low levels of IFN-gamma in human serum and plasma samples. The modifications in this protocol are easy to apply with basic laboratory equipment.

A simple in vitro technique for studies on induction of class II transplantation antigens on keratinocytes.

A new technique is described for evaluation of the induction and kinetics of class II transplantation antigens on keratinocytes. By culturing small rat or human skin specimens for 2-5 days in media containing gamma-interferon, class II antigens were detected on keratinocytes by immunohistochemistry. This technique is rapid and technically simple compared to keratinocyte cultures and raises the possibility of studying other cells in the skin.

Antiviral activity appearing in serum of renal transplant recipients. Its possible relation to immunological rejection.

Sera collected from a majority (15/16) of recipients of renal transplants occasionally contained low, but significant, levels of antiviral activity, maximally corresponding to 25-50 units of interferon-alpha per milliliter of serum in a bioassay. The exception was the only transplanted patient with a known cytomegalovirus infection, who demonstrated persistent high levels of antiviral activity corresponding to 200-400 interferon units/ml. Only the latter antiviral activity was sufficiently high for a partial characterization. It was pH 2 labile, did not have antigenic properties of interferon-alpha or beta, and may correspond to the lymphokine interferon-gamma. In consecutive serum samples from individual patients, the peaks of the antiviral activity occurred preponderantly in connection with clinical signs and consequent treatment of graft rejections. Interferon may, therefore, be a useful marker (at the serum level) of incipient graft rejection, and of certain viral infections, at least, provided that a more rapid sensitive and precise assay of this lymphokine is developed.

Detection of serum interferon-alpha by dissociation-enhanced lanthanide fluoroimmunoassay. Studies of patients with acute viral and bacterial infections.

A sensitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) was evaluated for ability to detect interferon-alpha (IFN-alpha) in serum of patients with acute infectious disease of less than one week's duration and a fever of > 38 degrees C. None of 36 patients with confirmed or probable bacterial disease was IFN-alpha positive. In contrast, 13/26 patients with viral infections had detectable levels of IFN-alpha in serum, all clearly positive (> or = 10 U/ml). The IFN-alpha positive serum samples were obtained early after onset of clinical disease, after a mean of 2.4 days. The IFN-alpha positive samples were obtained from 10 of the 12 patients with influenza or flu-like infection, and 3 of the 5 patients with varicella or herpes zoster. The IFN-alpha negative patients with viral disease (n = 9) included five patients with mononucleosis. The DELFIA should be useful in further studies of the value of IFN-alpha determinations in the identification of acute viral infections.

In HIV-1-infected patients, plasma levels of HIV-1 RNA are inversely correlated with IFN-alpha responsiveness of whole-blood cultures to sendai virus.

BACKGROUND: A diminished or totally blocked IFN-alpha production in cells from HIV-1-infected patients has been reported. OBJECTIVE: To investigate the relationship between the decreased in vitro production of IFN-alpha and the plasma level of HIV-1 RNA. STUDY DESIGN: Whole blood samples of 39 healthy subjects and 44 HIV-1-infected patients were incubated in the presence of Sendai virus for 24 h. IFN-alpha contained in supernatants was assayed by using an immunochemical method (DELFIA) and by using an antiviral assay. Plasma HIV-1 RNA was measured by the Amplicor HIV-1 monitor test. RESULTS: The levels of IFN-alpha obtained were significantly lower in cultures from HIV-1 infected patients than in control subjects (P<0.0001). The antiviral activity in supernatants of Sendai virus-activated whole-blood cultures, assayed by protection of MDBK cells against vesicular stomatitis virus (VSV), was significantly lower in cultures from HIV-1 infected patients than in corresponding controls (P<0.0001). IFN-alpha values determined by DELFIA and those determined by bioassay were significantly correlated. In vitro production of IFN-alpha by whole-blood cultures correlated well with the plasma levels of HIV-1 RNA (P<0.001). CONCLUSIONS: In HIV-infected patients an increased rate of HIV-1 replication is associated with reduced responsiveness to induction of IFN-alpha by indicator virus, suggesting that HIV-1 replication causes impaired production of IFN-alpha by blood cells or vice-versa.

Th1 and Th2 cytokine responses after measles antigen stimulation in vitro in bone marrow transplant patients: response to measles vaccination.

In seronegative autologous bone marrow transplanted (ABMT) patients, a sustained cell-mediated immunity (CMI) has been shown to impair the antibody response after measles vaccination. To investigate if this might be caused by a preferential Th1 cytokine response, interferon (IFN)-gamma and interleukin (IL)-10 production of peripheral blood mononuclear cells (PBMC) was analyzed after measles antigen (M-ag) stimulation in vitro. The non-specific immune response was measured by IFN-alpha, and IL-12 analyses. Fifty non-vaccinated patients following ABMT or allogeneic bone marrow transplantation (BMT) were included. IFN-gamma production was significantly higher in patients with a retained CMI to measles than in patients without (2.3 vs 0.8 IU/ml; P = 0.01). Only a non-significant tendency was seen in IL-10 production (48.6 vs 26.7 pg/ml; NS), whereas no difference was found in IFN-alpha or IL-12 production. A positive correlation between IFN-gamma and IL-10 production was found (r(s) = 0.49; P < 0.001). After vaccination of 14 ABMT children, there was an increase in PBMC IFN-gamma production in vitro (2.5 vs <0.1 IU/ml; P < 0.05), whereas no changes were seen in the IL-10, IFN-alpha, or antibody levels. These results suggest that both Th1 and Th2 cytokine production are increased by M-ag stimulation in patients with a retained CMI to measles, but the Th1 response seems to be stronger. The preferential Th1 stimulation and increase in IFN-gamma production after vaccination may lead to a reduction in the humoral immune response which may explain the negative correlation between antibody production and T cell reactivity prior to vaccination.

Induction of MxA mRNA in patients with neuroendocrine tumors after interferon treatment. Lack of correlation with antitumor response.

We have investigated the possibility of using induction of MxA mRNA in patients with neuroendocrine tumors undergoing interferon-alpha(IFN-alpha) treatment as a predictive test for antitumor effect. A total of 122 patients with various types of neuroendocrine tumors were included in the study. Blood samples were drawn 12 hours after administration of either recombinant or natural IFN-alpha for analysis of induction of MxA mRNA in peripheral blood leukocyte (PBLs). Total RNA was isolated and slot-blot hybridization was performed using a MxA cDNA fragment as a probe. All patients displayed induction of MxA mRNA. Out of 13 untreated patients, 4 had MxA mRNA as well as 2 out of 11 patients treated with somatostatin analogue. All IFN-alpha treated patients showed induction of MxA mRNA and there was no difference between patients demonstrating partial remission, stable disease or progressive disease. Moreover, there was no difference between recombinant or natural leukocyte IFN-alpha in the ability to induce MxA mRNA. Six patients developed neutralizing IFN-alpha antibodies with 1 patient presenting a tire of 3200 NU/ml. Development of neutralizing antibodies did not abrogate the induction of MxA mRNA, but in 3 patients with high antibody titres the antitumor effect was lost. We therefore conclude that IFN-alpha is able to induce MxA mRNA in PBLs from patients with neuroendocrine tumors but we could not find any correlation with the therapeutic outcome. Furthermore, development of neutralizing IFN-alpha antibodies, although abrogating the antitumor effect, might not block the antiviral activity.