Presence of cutaneous interferon-alpha producing cells in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) patients have increased levels of interferon-alfa (IFN-alpha) in the circulation but a reduced number of functionally intact natural IFN-alpha producing cells (IPC) in peripheral blood. In search for tissue localisation of activated IPC, we investigated skin biopsies from SLE patients for the occurrence of such cells. Eleven SLE patients with inflammatory skin lesions and six healthy controls were biopsied. An immunohistochemical technique (IH) and in situ hybridisation (ISH) were used to detect intracellular IFN-alpha protein and IFN-alpha mRNA, respectively. In all 11 biopsies from SLE lesions, a high number of IPC were detected by IH. In the nonlesional SLE biopsies we could also demonstrate IPC in 10/11 patients. In 6/11 SLE patients, IFN-alpha mRNA containing cells could be detected in the specimens. A low number of IPC were detected in 1/6 healthy controls by IH, but no ISH positive cells were seen. Our results demonstrate that SLE patients have active IPC in both dermal lesions and in noninflammatory skin. A recruitment of IPC from blood to peripheral tissues may explain the low number of circulating natural IPC in SLE patients. Because the type I IFN system is involved in the SLE disease process, these results are of interest for the understanding of the pathogenesis in SLE.

Patients with systemic lupus erythematosus (SLE) have a circulating inducer of interferon-alpha (IFN-alpha) production acting on leucocytes resembling immature dendritic cells.

Patients with active SLE often have an ongoing production of IFN-alpha. We therefore searched for an endogenous IFN-alpha-inducing factor (IIF) in SLE patients and found that their sera frequently induced production of IFN-alpha in cultures of peripheral blood mononuclear cells (PBMC) from healthy blood donors, especially when the PBMC were costimulated with the cytokines IFN-alpha2b and granulocyte-macrophage colony-stimulating factor (GM-CSF). The phenotype of the IFN-alpha-producing cells (IPC) as determined by flow cytometry corresponded to that of the natural IPC, resembling immature dendritic cells. The IIF activity in SLE sera was sometimes as high as that of a virus and was present especially in patients with active disease and with measurable IFN-alpha levels in serum. The IIF had an apparent molecular weight of 300-1000 kD and appeared to consist of both immunoglobulin and DNA, possibly being immune complexes. This endogenous IFN-alpha inducer may be of pathogenic significance, since a reported occasional adverse effect of IFN-alpha therapy in patients with non-autoimmune disorders is development of anti-dsDNA antibodies and SLE.

Patients with systemic lupus erythematosus have reduced numbers of circulating natural interferon-alpha- producing cells.

Systemic lupus erythematosus (SLE) patients often have continuous production of interferon-alpha (IFN-alpha), but production of in vitro IFN-alpha by peripheral blood mononuclear cells (PBMC) may be varyingly reduced. We here report that IFN-alpha production induced by Herpes simplex virus (HSV) in PBMC resembling immature dendritic cells and designated natural IFN-alpha producing cells (NIPC), was much more affected than that induced by sendai virus (SV) in monocytes. At the cell level, the frequency of HSV-activated NIPC was reduced 70-fold, but residual NIPC produced normal amounts of IFN-alpha (1-2 U/cell). The NIPC frequency increased 10-fold in SLE-PBMC, but not in control PBMC, when co-stimulated by the combination IFN-alpha-gamma and GM- CSF. No spontaneous IFN-alpha production by PBMCs was detected in SLE patients. While no SLE serum factor inhibiting IFN-alpha production was seen, sera of four out of 11 SLE patients induced IFN-alpha production in healthy control PBMC. We propose that the number of NIPC in SLE are reduced in blood because of recruitment to tissues and activation by an endogenous IFN-alpha inducer, as well as because of lack of co-stimulatory cytokines. IFN-alpha produced in SLE could be of pathogenic significance, because autoimmune diseases develop in patients with infections or tumours during IFN-alpha therapy.

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.

The cell surface phenotype of human natural interferon-alpha producing cells as determined by flow cytometry.

The relationship between the so-called natural interferon-alpha (IFN-alpha) producing cell (IPC), stimulated to produce IFN-alpha by herpes simplex virus type 1 (HSV), and of dendritic cells (DC) in peripheral blood leucocytes was investigated. The simultaneous expression of cell surface antigens and intracellular IFN-alpha in the HSV-stimulated IPC (HSV-IPC) was examined by flow cytometry (FCM). The HSV-IPC were infrequent, < 0.3% of the mononuclear leucocytes, and with homogeneous light scatter characteristics. The HSV-IPC were confirmed to lack leucocyte lineage specific markers, and to express CD4, CD36 and HLA-DR. Furthermore, they expressed high levels of CD44, CD45RA and CD45RB, and lower levels of CD40, CD45R0, CD72 and CD83. The HSV-IPC expression of CD13, CD33 and Fc epsilon RI were weak but significant, while no CD5, CD11b, CD16, CD64, CD80 or CD86 were detected. Sorted pure HSV-IPC had irregular shaped nuclei, many mitochondria and vesicles, and rugged cell membranes without veils. Sorted HSV-IPC stimulated proliferation of autologous T cells from HSV immune donors. Thus, the HSV-IPC in many respects resemble immature DC, but clearly differ from typical mature DC. However, they may also represent a specialized population of efficient IFN-alpha producing cells.

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.

Lectins inhibit the Aujeszky's disease virus-induced interferon-alpha production of porcine peripheral blood mononuclear cells.

The interaction between virus and peripheral blood mononuclear cells (PBMC) required to elicit the production of interferon-alpha (IFN-alpha) by the so-called natural interferon-producing cell is unknown. However, results from inhibition experiments suggest that viral glycoproteins are essential in this IFN induction process. We demonstrate here that cellular glycoproteins also appear to be involved in the initiation of IFN-alpha production. Lectins, that is, sugar binding glycoproteins, inhibited the Aujeszky's disease virus-induced IFN-alpha production of porcine PBMC by up to 99%. The level of inhibition varied with lectin used (concanavalin A, Galanthus nivalis lectin, Helix pomatia lectin, and lentil lectin). Preincubation experiments with porcine cells and concanavalin A (ConA) revealed that the lectin exerted its major effect directly on the PBMC. Although the IFN-alpha production in some cases was reduced by more than 90%, the PBMC were still able to proliferate in response to mitogenic lectins. The ConA-mediated inhibition of the IFN-alpha production was reduced if the lectin was added later than 6-8 h after the start of induction and was not mediated by soluble factors. Both orthovanadate and staurosporine inhibited the IFN-alpha production and did not relieve the ConA-mediated inhibition. Thus, ConA seems to interfere with the early events during IFN-alpha induction, but the mechanisms behind this interference could not be clarified.

Interferon system defects in malignant T-cells.

Deletions of chromosome 9p21-22 occur in acute lymphocytic leukemia (ALL), melanoma and glioma. With some exceptions, these deletions include the alpha- and beta-interferon (IFN) genes. In this study, the frequency of alpha- and beta-IFN gene deletions was investigated in 17 T-cell lines, and losses of IFN genes were related to other aspects of the IFN system. Deletions of alpha-/beta-IFN genes were observed in 7/17 cell lines. In two cases the deletions were homozygous for both loci. In most cases aberrations of chromosome 9 were also apparent on cytogenetic analysis. An increased proportion (40% as compared to the expected 13%) of the remaining ten cell lines showed homozygosity for all five common polymorphic alpha-/beta-IFN markers, possibly implicating allelic deletion by loss of heterozygosity (LOH) in some of these clones. The cell lines showed a large variability in IFN production, IFN-alpha receptor number, susceptibility to IFN measured as induction of the enzyme 2',5' oligoadenylate synthetase and cell growth inhibition. No correlations between loss of IFN genes and IFN-producing capacity, or susceptibility to IFN, were found. Of the seven cell lines with a normal IFN-gene dosage and heterozygosity for the alpha- and beta-IFN genes, three had a deficiency in their IFN-producing capacity and one was also insensitive to growth inhibition by IFN. All IFN-producing cell lines predominantly produced beta-IFN.

Age-related increase of porcine natural interferon alpha producing cell frequency and of interferon yield per cell.

Porcine blood mononuclear cells (PBMC) were shown to secrete interferon alpha (IFN-alpha) after induction by a coronavirus, the transmissible gastroenteritis virus (TGEV). IFN-alpha producing cells, referred to as natural interferon alpha producing (NIP) cells, were detected by an ELISPOT assay using anti-porcine IFN-alpha monoclonal antibodies. The frequency of NIP cells among blood cells is low, at most 40-110 per 10(5) PBMC and each NIP cell was found to produce several units of IFN. We have shown that NIP cell frequency and IFN yield per cell gradually increased with the age of the donor animals, from the neonatal period to the adult age, with a significant increase around puberty. Our present results also indicate that NIP cells may be influenced by physiological and genetic factors; thus (1) NIP cell frequency and IFN yield per cell were decreased during lactation; (2) Chinese (Meishan) pigs were found to have higher NIP cell frequency and IFN yield per cell than European (Large White) animals.

The leukocyte function-associated antigen-1 (LFA-1) is involved in the interferon-alpha response induced by herpes simplex virus in blood leukocytes.

The role of the leukocyte function-associated antigen-1 (LFA-1) family of integrins (beta 2 integrins) in the interferon-alpha (IFN-alpha) response was examined, using human peripheral blood mononuclear cells (PBMCs) stimulated in vitro by glutaraldehyde-fixed Herpes simplex virus-infected WISH amnion cells. Monoclonal antibodies (mAbs) to the beta 2 chain (CD18) and to the alpha chain of LFA-1 (CD11a) reduced the number of IFN-alpha-producing cells (IPCs) by 30-50%, but mAbs to CD11b or c caused no inhibition. The IB4 mAb to CD18 was inhibitory when added during the first 2 h of the IFN-alpha response, but did not alter its kinetic. In contrast, the IB4 prevented the early enhancement of the IFN-alpha response caused by addition of interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). However, a delayed down-regulation of the IPC response occurred in such PBMC cultures, and a paradoxical increase in the total production of IFN-alpha. The results suggest that LFA-1 (CD11a/CD18) participates in the early phase of the IFN-alpha response and may be activated by cytokines such as IL-3 and GM-CSF.

Interferon-alpha but not -beta genes require de novo protein synthesis for efficient expression in human monocytes.

Monocytes produce interferon-alpha (IFN)-alpha) and -beta when human peripheral blood mononuclear cells (PBMCs) are stimulated in vitro by Sendai virus (SV). We found that about 70% of the IFN-producing cells (IPCs) expressed both IFN-alpha and -beta mRNA; the rest expressed only IFN-beta mRNA. In the presence of the protein synthesis inhibitor cycloheximide (CHX), the frequency of IFN-alpha mRNA-containing cells, measured after 6h, was decreased by 85-90%. Results of nuclear run-on transcription assays showed that CHX inhibited IFN-alpha gene expression. The frequency of IFN-beta mRNA-containing cells was not reduced by CHX. Actually, a threefold increase was observed at the lower CHX concentrations. Studies on the kinetics of IFN-alpha/beta mRNA induction showed that CHX accelerated the appearance of IFN-beta mRNA-containing cells, increased IFN-beta mRNA levels, and delayed the normally occurring post-inductional decrease of IFN-beta mRNA. Unexpectedly, an initially normal or even accelerated IFN-alpha mRNA response was seen in the presence of CHX during the first 3-4 h after SV stimulation. This occurred in a small proportion of the potential IPCs. However, CHX prevented the subsequent marked increase of IFN-alpha mRNA levels. Preincubation of PBMCs for 6 h in conditioned medium (CM) containing IFN and other cytokines prevented the CHX-mediated inhibition of IFN-alpha mRNA. Without preincubation this was not seen. The preincubation in CM caused an accelerated appearance of IFN-alpha mRNA, resembling that of IFN-beta mRNA. The results suggest that IFN-alpha and -beta genes are differentially regulated in the same monocytes, the former requiring de novo synthesis of intracellular protein(s) for efficient expression.

The induction of interferon-alpha and interferon-beta mRNA in human natural interferon-producing blood leukocytes requires de novo protein synthesis.

The induction of interferon-alpha (IFN-alpha) and IFN-beta mRNA in natural IFN producing (NIP) cells in cultures of human peripheral blood mononuclear cells (PBMCs), stimulated by glutaraldehyde-fixed Herpes simplex virus type 1 (HSV)-infected WISH cells, was studied. The protein synthesis inhibitor cycloheximide (CHX) totally prevented the appearance of both IFN-alpha and IFN-beta mRNA, also in cultures supplemented with a conditioned medium (CM) assumed to contain soluble factors necessary for the IFN induction. However, when PBMCs were preincubated for 4 h in medium supplemented with fetal bovine serum (FBS) with or without addition of CM, the subsequent induction of IFN-alpha/beta mRNA became partially resistant to CHX. In serum-free medium containing interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF), the early induction of IFN-alpha mRNA became resistant to CHX, and, in contrast to FBS and CM supplemented medium, this was observed also without a preincubation of the PBMCs. In contrast, IL-1, IL-2, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha), IFN-alpha, or IFN-gamma had no such effects. Our results suggests that de novo synthesis of proteins normally is required for the induction of IFN-alpha/beta mRNA. Such proteins might be cytokines, possibly CSFs, which in turn also may require protein synthesis for their actions. In contrast, the actual triggering signal provided by the HSV-inducer is independent of protein synthesis.

Interferons and the colony-stimulating factors IL-3 and GM-CSF enhance the IFN-alpha response in human blood leucocytes induced by herpes simplex virus.

Human peripheral blood mononuclear cells (PBMC) were stimulated to produce interferon-alpha (IFN-alpha) by glutaraldehyde-fixed Herpes simplex virus type 1 (HSV)-infected WISH amnion cells in vitro. Different cytokines were included during the stimulation and tested for their ability to enhance the IFN-alpha response which occurs in the natural IFN-alpha producing (NIP) leucocytes. The total production of IFN-alpha and the numbers of IFN-alpha producing cells (IPCs) were increased by interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Their most marked effect was to reduce the time required for induction of the IPC by the HSV-infected cells, thereby causing both an earlier peak of IPC numbers and secretion of IFN-alpha. Addition of IFN-alpha 2b did not alter the kinetics of the IFN-alpha response in the same way as the two CSFs, but instead generally increased the IPC numbers and the production of IFN-alpha. The IL-3 and GM-CSF, especially in combination with IFN-alpha, had the most pronounced enhancing effects on IPC numbers when PBMC were induced at low cell concentrations. The cytokines IL-1, IL-2, IL-4, IL-6 or tumour necrosis factor-alpha (TNF-alpha) had no detectable effects on the IFN-alpha response. The results suggest that cytokines such as the CSFs and IFNs may be involved in the regulation of NIP cell functions.

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.

Infrequent but efficient interferon-alpha-producing human mononuclear leukocytes induced by herpes simplex virus in vitro studied by immuno-plaque and limiting dilution assays.

Human blood mononuclear leukocytes (PBMCs) producing interferon-alpha (IFN-alpha) after stimulation by herpes simplex virus type 1 (HSV) in vitro were identified by a filter immuno-plaque assay. Individual IFN-alpha-producing cells (IPCs) yielded between 0.5 and 2 units IFN-alpha, sufficient to protect cultures of MDBK cells against a viral challenge. Therefore, their frequency could be determined by a limiting dilution assay as well as by an immuno-plaque assay. Similar estimates of between 2 and 55 IPCs per 10(4) PBMCs at the peak of the IFN-alpha response were obtained by the two methods. IPCs were first detected 3 h after stimulation by HSV; their number peaked at 8 h and then declined. IPC frequencies were influenced by the concentrations of HSV and PBMCs during induction, but the quantity of IFN-alpha produced per IPC was relatively constant. The relation between the numbers of IPCs and PBMCs was linear at high PBMC concentrations, whereas at low PBMC concentrations fewer IPCs than expected were detected. The response could be fully restored by adding a combination of filler cells (Namalwa or U937 cells) and conditioned medium from 6-h HSV-induced PBMC cultures. Our results suggest that HSV induces an IFN-alpha response in a relatively rare population of efficient IPCs by complex mechanisms, which may involve cell cooperation and/or production of soluble factors.

Deficient herpes simplex virus-induced interferon-alpha production by blood leukocytes of preterm and term newborn infants.

The ability of peripheral blood mononuclear cells (PBMC) from newborn infants, gestational age 24-42 wk, to produce interferon-alpha (IFN-alpha) on the first day after birth was studied in vitro. Human amnion cells (WISH) coated with herpes simplex virus type I and fixed by glutaraldehyde were used as IFN-alpha inducers. Individual IFN-alpha producing cells (IPC) among PBMC were determined by an immunoplaque assay. The frequency of IPC was low in all premature (less than or equal to 36 wk) infants (median 0.3 IPC/10(4) PBMC, range 0.0-2.6), and significantly higher (median 2.0 IPC/10(4) PBMC, range 0.0-16.4) in term infants (greater than 37 wk). The frequencies were lower in both groups of infants than in adults (7.3 IPC/10(4) PBMC, range 2.0-23.7). When a conditioned medium from cultures of herpes simplex virus type I-stimulated PBMC from adults was added to the IFN induction cultures, the frequencies of IPC increased in PBMC from both preterm and term infants, and in the latter group did not differ significantly from adult levels. The median production of IFN-alpha per IPC was 1.1 U (range 0.0-2.8) in premature infants, 1.0 U (range 0.0-8.8) in term infants and 3.2 U (range 1.5-8.0) in adults. When concentrations of PBMC in the cultures [corrected] were decreased, a decline of IPC frequencies occurred. This decline was more marked and started at higher PBMC concentrations in infants than in adults, and was prevented by addition of conditioned medium from herpes simplex virus type I-stimulated cultures of PBMC from adults.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of herpes simplex virus-induced alpha interferon-secreting human blood leucocytes by a filter immuno-plaque assay.

A filter immuno-plaque assay was developed which detects alpha interferon (INF-alpha)-secreting human peripheral blood leucocytes (PBL). Polyclonal anti-IFN-alpha antibodies were fixed to the nitrocellulose membrane bottoms of 96-well Millititer plates, which also contained monolayers of glutaraldehyde-fixed human WISH amnion cells infected by Herpes simplex virus type 1 (HSV). Such cells are potent IFN inducers and during a 16 h cocultivation with PBL, IFN-alpha was absorbed by the membrane-bound polyclonal antibodies around IFN-alpha-secreting cells. This IFN-alpha was detected with murine monoclonal antibodies against IFN-alpha and peroxidase-labelled antibodies against murine immunoglobulin, using diaminobenzidine as substrate. Distinctly stained plaques were seen, the frequency of which gave a minimal estimate of approximately 10 IFN-alpha-producing cells in 10(4) PBL (range in 12 blood donors 2.95-25.1). Fewer plaques than expected were seen at low PBL numbers per culture, one explanation being that cell interactions then limit the IFN-alpha response. The immuno-plaque assay should be useful in further studies of the cellular basis of the IFN-alpha response.