Setting the scene: a historical and personal view of immunologic diseases, autoimmunity and ANA.

Dr. Friou reviews here his research experiences in immunology beginning over fifty years ago, and dealing with his early background experience with group A streptococcus, antihyaluronidase, and rheumatic fever, and cell mediated responses in Boeck's sarcoid. Details of his introduction of the immunofluorescent technique of Harvard Prof. Albert Coons into the SLE-ANA area, with the complexities and difficulties of early involvement with the technique are described. The simple, but extremely clever experiment of Prof. Miescher of Geneva which created an ideal situation for the application of immunofluorescence to the SLE area is described. Technical details of the earliest immunofluorescent ANA studies are presented, together with firm data that these early studies, which included many human sera and studies of the nature of the antibody target, had been completed by February 21, 1957, well in advance of any other laboratory. Important subsequent related studies from international laboratories are reviewed.

Clinical and biological characteristics of Ureaplasma urealyticum induced polyarthritis in a patient with common variable hypogammaglobulinaemia.

Persistent infectious polyarthritis caused by Ureaplasma urealyticum in a patient with common variable hypogammaglobulinaemia is described. The patient developed a symmetrical, destructive polyarthritis and tenosynovitis associated with a markedly depressed synovial fluid glucose concentration and characteristic soft tissue abscesses. The ureaplasma organism developed resistance to multiple antibiotics and persisted for five years. The organism was identified repeatedly in many joints by culture, confirmed by DNA hybridisation, and mycoplasma-like structures were shown in synovial tissues by electron microscopy.

Effects of human 17 kDa interleukin 1, 25-31 kDa thymocyte stimulating activity and the 6-9 kDa interleukin 1 inhibitor on calcium release in the newborn murine calvarial assay.

The effects of human monokines on calcium release from cultured newborn murine calvarium were studied. Highly purified interleukin 1 (IL-1) (17 kDa) and recombinant IL-1 beta in the concentration range 0.2-20 U/ml released significant amounts of calcium. Mean resorption indices (RI) at 0.2 U/ml were 1.28 and 1.49, and at 20 U/ml, were 1.82 and 1.72, respectively. Calcium release was abrogated by the cyclooxygenase inhibitor piroxicam. Thymocyte stimulating activity (TSA) 25-31 kDa alone at 0.14 U/ml released calcium in a prostaglandin dependent manner with a mean RI of 2.13, a significantly greater calcium release than that obtained by 17 kDa IL-1 at 20 U/ml. The 6-9 kDa inhibitor of IL-1 induced thymocyte proliferation alone also released calcium in a prostaglandin dependent manner with a mean RI of 2.29 at 200 inhibitory U/ml. Addition of 6-9 kDa IL-1 inhibitor to the 25-31 kDa material did not significantly change the calcium release, whereas addition of the inhibitor to 17 kDa IL-1 produced a significant increase in calcium release.

Changes in circulating monocytes in patients with progressive systemic sclerosis.

Circulating monocytes in 30 patients with progressive systemic sclerosis (PSS, scleroderma) and 28 age and sex matched normal controls were studied. Binding of the lectin peanut agglutinin (PA) was significantly reduced in PSS monocytes (p less than 0.001) together with a reduction in the density of nonspecific esterase staining (p less than 0.001) suggesting advanced maturation. Using monoclonal antibodies to identify cell surface markers, we demonstrated a significant reduction in PSS monocytes bearing the Leu M2 antigen (Mac 120, antigen presenting cells) over controls (p less than 0.05), but were unable to show any differences in the monocyte subpopulations using antisera against Leu M3 and HLA-DR surface antigens. The ectoenzymes 5'-nucleotidase (5'N) and alkaline phosphodiesterase 1 (APD1) were lower and leucine aminopeptidase (LAP) levels were higher in patients with PSS, compatible with immune activation. Interferon-gamma levels in serum did not appear to account for these changes, whereas the levels of Clq binding complexes correlated inversely with the levels of LAP (p less than 0.05). There was a strong correlation between the number of Leu M3 positive cells and the level of the ectoenzyme LAP (p less than 0.001). With increasing disease duration, higher levels of Clq binding complexes were detected (p less than 0.05). These results indicate that monocytes in PSS differ from those in normals and appear to have undergone advanced differentiation and activation changes.

Interleukin 1 inhibitor masks high interleukin 1 production in acquired immunodeficiency syndrome (AIDS).

Monocyte functions, including interleukin 1 (IL-1) production, have been shown previously to be impaired in acquired immunodeficiency syndrome (AIDS). We have fractionated culture supernatants from unstimulated peripheral blood mononuclear cells (PBMCs) to determine whether the low IL-1 activity in AIDS was due to the presence of IL-1 inhibitors. The results demonstrate that PBMCs from patients with AIDS produce increased amounts of IL-1 activity compared with those of controls together with marked increases (10- to 20-fold) in the amounts of 50,000-100,000 and 6000-9000 molecular weight (MW) factors which inhibit IL-1 activity. These inhibitors mask IL-1 activity measured in the standard thymocyte proliferation assay for IL-1. The 6000-9000 MW IL-1 inhibitor shows the greatest increase in all AIDS patients (n = 5) compared with that of controls (n = 7). This inhibitor may block the IL-1 dependent maturation of T lymphocytes in AIDS and thereby contribute to the immunodeficiency.

Increased production of an interleukin 1 (IL-1) inhibitor with fibroblast stimulating activity by mononuclear cells from patients with scleroderma.

We have previously demonstrated low IL-1 activity produced by peripheral blood mononuclear cells (PBMC) from patients with scleroderma (Sandborg et al., 1985) and the production of a 6-9 K IL-1 inhibitor by normal monocytes (Berman et al., 1986). To determine whether this inhibitor accounted for the low IL-1 activity present in scleroderma, the production of IL-1 and IL-1 inhibitor by PBMC from eight scleroderma patients was studied. Concentrated supernatants from 24 h cultures of unstimulated PBMC were fractionated on Sephacryl S-200 and tested for IL-1 and IL-1 inhibitor activity in the standard IL-1 thymocyte proliferation assay. In seven of eight patients, IL-1 inhibitor production was increased (average 3.3 X) compared to matched controls. IL-1 production was less than controls in six of eight patients. Partially purified preparations of the 6-9 K mol. wt IL-1 inhibitor were inhibitory to IL-1 induced thymocyte proliferation but stimulatory to fibroblast proliferation when purified by gel chromatography and chromatofocusing (pI 4.5-5.6). These data suggest that an IL-1 inhibitor with fibroblast stimulating activity is produced in higher amounts by PBMC from patients with scleroderma, and may contribute to the fibroblast proliferation and excessive collagen synthesis which is typical of this disease.

Immune complexes in synovial fluid and serum from patients with disseminated gonococcal infection: evidence for local immune complex formation within the joint.

Twenty one patients with acute arthritis associated with disseminated gonococcal infection (DGI) were studied. Synovial fluid (SF) from 14 and serum from 15 (matched in eight) were assayed for the presence of immune complexes (IC) by the Raji cell immunofluorescent assay (Raji IFA) and the 125I-Clq polyethylene glycol (PEG) binding assay. Higher levels and frequency of IC were detected in the SF by both IC assays and these were associated with a significant increase in complexes containing IgM over serum (p less than 0.02). Complexes containing IgG were found predominantly in serum and were infrequent in SF (p less than 0.003). These data suggest that the arthritis of DGI may result from primary immune complex formation within the synovial cavity after local antibody synthesis within the synovium in response to gonococcal seeding.

Studies of an interleukin 1 inhibitor: characterization and clinical significance.

Supernatants from 24 h cultures of human peripheral blood mononuclear cells (PBMNC) were fractionated and tested for interleukin (IL-1) activity in the mouse thymocyte assay with phytohaemagglutinin (PHA). By the addition of individual supernatant fractions together with partially purified IL-1 to the thymocyte assay, we demonstrate the presence of strong inhibitory activity with a mol. wt of 5,000-9,000 and an isoelectric point of 4.5-5.6. The activity is both heat (56 degrees C) and acid (pH 1.5) resistant. This inhibitor has no detectable suppressive effect on optimal and suboptimal concanavalin A (Con A), pokeweed mitogen (PWM), and PHA responses of PBMNC. The action of the inhibitor appears to be specifically directed against IL-1 action on thymocytes and has no inhibitory effect on interleukin 2 (IL-2) activity. The findings show that adherent PBMNC produce both IL-1 and a factor which opposes IL-1 action on thymocytes but not on peripheral (mature) T cells. This factor may regulate T cell maturation, activation, and proliferation.

Loss of epidermal Langerhans' cells and endothelial cell HLA-DR antigens in the skin in progressive systemic sclerosis.

Skin biopsies from the volar aspect of the forearm were studied in 26 patients with progressive systemic sclerosis (PSS) (16 diffuse, 10 CREST) and 4 controls using monoclonal antibodies against Langerhans' cells, T lymphocytes, macrophages, B lymphocytes, NK/K cells and HLA-DR antigen(s). Langerhans' cells were reduced or absent (anti-T6, anti-HLA-DR) in 19 of 20 clinically involved and in all 6 uninvolved PSS skin biopsies. Electron microscopic studies of 3 PSS patients indicated a reduction in the number of Langerhans' cells, with normal morphology of the remaining. HLA-DR antigen(s) on dermal endothelial cells were absent or reduced in 8 of 20 involved and 5 of 6 uninvolved PSS skin biopsies, but were present on the surface of dermal mononuclear cells presumably representing activated T lymphocytes. Increased numbers of dermal macrophages were found in 19% of PSS biopsies compared with controls. Absence of Langerhans' cells appears to represent the most widespread immunopathological feature of PSS. It is also associated with absent endothelial HLA DR surface antigens and activated T lymphocytes within the dermis.

Interleukin-1 production by mononuclear cells from patients with scleroderma.

Interleukin-1 (IL-1) production by peripheral blood mononuclear cells (PBMC) from patients with scleroderma and healthy controls was studied. Supernatants from unstimulated PBMC cultures from 10 of 13 patients with progressive systemic sclerosis (PSS) had significantly less IL-1 activity as measured by thymocyte proliferation than controls. IL-1 activity per monocyte/macrophage in both patients and controls was 10 times greater when PBMC were cultured at 10(5) cells/ml compared to 10(6) cells/ml. Five-fold dilution of supernatants from PBMC cultured at 10(6) cells/ml revealed more IL-1 activity than undiluted supernatant and addition of indomethacin increased IL-1 activity primarily of the undiluted supernatant. The results show that IL-1 activity from crude PBMC supernatants from PSS patients is low and may be regulated by non-dialysable inhibitors produced by PBMC and/or cell interactions.

Circulating anti-tumor and autoantibodies in breast carcinoma: relationship to stage and prognosis.

Serum antibodies to breast tumor antigen(s) and circulating autoantibodies were tested in 175 patients with various stages of carcinoma of the breast, followed for a mean period of 51 months. Antibodies to surface membrane and to cytoplasmic antigens of autologous and allogeneic tumor cells were measured. Peripheral lymphocyte count and skin reaction to six recall antigens were also tested. Patients with metastatic disease had significantly lower prevalence of antibodies to autologous tumor cells and lower total lymphocyte count than patients with early breast cancer. Patients with locally advanced disease (greater than or equal to 4 positive axillary nodes) had the highest frequency of anti-tumor antibodies, the second highest lymphocyte count, but with the lowest prevalance of autoantibodies. Presence or absence of anti-tumor or autoantibody did not correlate with results of skin tests or other standard blood tests. Among patients with locally advanced or metastatic breast cancer, those who had a positive skin test or whose lymphocyte count was 1500 to 2500 per cu mm had significantly better 5-year absolute survival rates (p = 0.04, p = 0.002, respectively). This study suggests that in patients with locally advanced or metastatic breast cancers, skin test reactivity and optimal peripheral lymphocyte count may be useful prognostic indicators. In contrast, neither the presence of anti-tumor antibodies to membrane or cytoplasmic antigens, nor the presence of autoantibodies, correlates with prognosis in patients with early or late breast cancers.

In vitro effects of 4-hydroperoxycyclophosphamide on the morphology and function of human peripheral blood mononuclear phagocytic cells (macrophages).

Human peripheral blood mononuclear cells were incubated for 1 hr in 4-hydroperoxycyclophosphamide (0.5 to 10.0 micrograms/ml), and the adherent, esterase-positive cells (macrophages) were studied. At 2 hr, a reduction was noted in both latex particle ingestion and Fc gamma receptor binding and phagocytosis. At 24 hr, spreading and pinocytosis were reduced, and cytoplasmic vacuoles developed. This vacuolization represented dilatation of the rough endoplasmic reticulum. These morphological and functional changes occurred with 4-hydroperoxycyclophosphamide concentrations which did not reduce viability or produce detectable DNA alkylation. This effect on macrophages may offer a mechanism whereby low-dosage cyclophosphamide could modify the immune response.

Limulus assay for bacterial endotoxin in synovial fluid.

The limulus assay for the detection of bacterial endotoxin has been applied to the study of synovial fluid. Three of 5 patients (60%) with culture-positive gonococcal arthritis had positive SF limulus assay results; as did 2 of 11 (18%) with presumptive evidence of gonococcal arthritis, 3 of 6 (50%) with nongonococcal infectious arthritis, and none of 47 patients with noninfectious arthritis. Endotoxin levels ranged from 0.25 to 128.0 ng/ml. As at present applied to synovial fluid the commercial limulus assay appears to be specific for the infectious process but apparently lacks sensitivity.

Serum antibodies to human fetal antigens in patients with systemic lupus erythematosus (SLE).

Serum antibodies to human fetal antigens were measured by a radiolabeled anti-immunoglobulin binding assay by using human fetal fibroblasts (Flow cell line No. 1000) as target cells. High titers of IgG antibody to the fetal cells were found in sera of patients with systemic lupus erythematosus (SLE). The antibody reacted with surface membrane antigens shared by various fetal tissues of human and murine origin but not by adult tissues. The reaction of the SLE antibody to the fetal cells was inhibited by heterologous antiserum to the Flow 1000 cells and antiserum to murine embryonic fibroblasts, but not by antiserum to human alpha-fetoprotein or human fibronectin. Absorption of SLE serum with isolated nuclei did not abolish the reaction indicating that these were not anti-nuclear antibodies. The antibody activity was found to reside in the F(ab')2 fragment. The serum titer of the anti-fetal antibody was higher in SLE patients with active disease than those in clinical remission.

Ductular carcinoma of the breast: serum antibodies to tumor-associated antigens.

Serum antibodies to tumor-associated antigens of breast carcinoma have been studied by indirect immunofluorescence in 109 patients with breast carcinoma and 125 controls, including age/sex matched normal individuals, patients with nonmalignant disease, and patients with malignant disease other than breast cancer. We report here that sera of a large proportion of patients with ductular carcinoma of the breast have antibodies to cell surface and/or intracellular antigens of autologous tumor cells and include evidence that the antigens are absent from a considerable range of normal and other types of malignant tissues. In addition to testing of control sera, specificity of the reacting antibodies was investigated further by testing of sera with normal breast tissue and the absorption of sera from breast cancer patients with various normal tissues and cancer cells. The significance of the findings in breast cancer is discussed.

Complement fixing antibodies to DS-DNA in systemic lupus erythematosus: a study using the immunofluorescent Crithidia luciliae method.

Crithidia luciliae, a hemoflagellate, was used in an immunofluorescent procedure to assay for antibodies to ds-DNA and their capacity to fix complement. Positive reactions with this method were limited to systemic lupus erythematosus patients, and sensitivity was comparable to the DNA binding assay. Complement fixing activity of antibodies to ds-DNA in 45 sera was determined using kinetoplast ds-DNA of Crithidia luciliae and an antiserum to C3. Complement fixing antibodies to ds-DNA were found in nearly all patients with documented active renal involvement and absent in nearly all patients with no, or inactive renal involvement.

IgG antibodies to double-stranded DNA in systemic lupus erythematosus sera. Independent variation of complement fixing activity and total antibody content.

Antibodies to double-stranded deoxyribonucleic acid were studied using the kinetoplast of Crithidia luciliae. Titers were determined separately by conventional immunofluorescence and the complement fluorescent technique, and results by the two methods were compared. Complement fixing activity varied independently of antibody content in whole serum and in IgG fractions. The well established correlation of complement fixing activity of this antibody with activity of lupus nephritis appears related, therefore, to qualitative rather than solely quantitative differences. This finding has important implications for the clinical assessment of patients with lupus, and investigations on the relationship of anti-DNA antibodies to lupus nephritis.

Cytotoxicity in progressive systemic sclerosis: no evidence for increased cytotoxicity against fibroblasts of different origin.

Peripheral blood mononuclear cells from 13 patients with progressive systemic sclerosis and 10 normal controls were tested in a cytotoxicity assay against fibroblasts derived from normal adult skin, scleroderma-involved skin and fetal skin. No differences between controls and patients were demonstrated. Sera from patients and controls revealed no differences in cytotoxic effect on scleroderma fibroblast cultures, and patients' sera did not induce antibody-dependent cell-mediated cytotoxicity when added in combination with control peripheral blood mononuclear cells.