An abnormal adherence of monocytes to fibronectin in thyroid autoimmunity has consequences for cell polarization and the development of veiled cells.

Blood monocytes of patients with thyroid autoimmune disease (TAID) display defects in rearranging their cortical actomyosin cytoskeleton ('polarize') in response to chemoattractants. Such rearrangements also take place after the adherence of monocytes to the extracellular matrix (ECM). It is therefore not surprising that monocytes are primed after fibronectin (FN) adherence, displaying an enhanced polarization toward chemoattractants. We investigated the integrin expression and chemoattractant-induced polarization of monocytes of TAID patients before and after FN adherence. Since cytoskeletal rearrangements are also required during the transition of monocytes into veiled antigen-presenting cells (VCs), we investigated such transition of FN-adherent monocytes of TAID patients. Adherent and nonadherent monocyte populations from TAID patients and healthy controls were subjected to a polarization test with the chemoattractant fMLP (or MCP-1), FACS analyses (FITC-labelled FN, CD29, CD49e, d, b and a) and tested for their capability to develop into veiled APC. Monocytes of healthy individuals showed an improved chemoattractant-induced cell polarization after FN adherence, not reflected by TAID monocytes, in which chemoattractant-induced polarization worsened. Monocytes of healthy individuals up-regulated CD49e and d integrins and their capability to bind FITC-labelled FN after adherence to a FN-coated plate, as well as enhancing their capability to generate T cell-stimulatory VCs. Monocytes of TAID patients did not. These data indicate that integrin- (and chemokine-) mediated functions are hampered in monocytes of TAID patients. Because integrin action is pivotal to processes such as monocyte adherence to endothelial cells, uropod formation, migration into tissues and differentiation into APC and macrophages, these defects might underly immune dysbalances important in thyroid autoimmune development.

Homotypic cluster formation of dendritic cells, a close correlate of their state of maturation. Defects in the biobreeding diabetes-prone rat.

Aggregation of dendritic cells (DCs) in homotypic clusters has been described in vivo in lymph and skin, and here we report studies on homotypic clustering of rat splenic (s) DCs in vitro. Wistar rat sDCs readily formed homotypic clusters in culture, which increased in number and size over time (with a peak at t = 3 h). Keeping the cells at higher densities or treatment with anti-CD43 induced more and larger homotypic clusters. After such enhanced clustering the DCs had increased their T cell stimulating capabilities in syngeneic mixed lymphocyte reaction, and had a higher expression of CD80 and CD86 (signs of maturation). Ag transfer from bovine serum albumin-fluorescein isothiocyanate-pulsed to unpulsed DCs was observed during clustering. Here we also show that sDCs of the biobreeding diabetes-prone (BB-DP) rat, a model of autoimmune diabetes/thyroiditis, formed fewer and smaller clusters than Wistar sDCs, and that DC-DC clustering resulted in only a modest maturation of the cells (as determined in syn MLR and by phenotyping). Anti-CD43 completely restored the clustering defect BB-DP DCs in vitro, yet T cell-stimulating capability was only restored to a limited extent. Ag transfer in BB-DP DC clusters was similar.

Analysis of peripheral blood lymphocyte subsets, NK cells, and delayed type hypersensitivity skin test in patients with premature ovarian failure.

PROBLEM: Premature ovarian failure (POF) probably belongs to the group of autoimmune endocrinopathies. Cell-mediated immune parameters were investigated. Sex steroids have a profound effect on the immune system. POF patients and postmenopausal control women (PM) were tested with or without estrogen substitution. METHOD: A novel FACS analysis system (using double labeling techniques) was used in 30 patients with POF to enumerate the subjects of peripheral blood lymphocytes and NK cells. Eighteen PM women and 30 healthy men and women served as controls. We also tested the delayed type hypersensitivity skin test (DTH) toward Candida in the POF patient group to be informed on their cell-mediated immune function. RESULTS: The numbers of blood lymphocytes, CD3+, CD4+ and CD8+T cells, were not abnormal in POF patients. However, HLA-DR+T cells were increased in POF patients and in PM women (P < 0.05). These elevated numbers were partially reversible by estrogen substitution. The number of CD19+ cells (B cells) was elevated, whereas CD3-/CD16+/CD56+ cells (NK cells) were decreased in POF patients (P < 0.05), irrespective of estrogen substitution. DTH skin tests toward 0.1% Candidin (0.1 ml intradermal injection) were negative in 11 out of 20 tested POF patients, compared to only 2 out of 10 tested controls (P < 0.05). CONCLUSION: POF patients show numerous immune cell abnormalities. These abnormalities were only partially due to estrogen deficiency. We hypothesize that these abnormalities either lead to ovarian autoimmunity or may have direct effects on the ovarian function.

Effect of thyroid hormones and other iodinated compounds on the transition of monocytes into veiled/dendritic cells: role of granulocyte-macrophage colony-stimulating factor, tumour-necrosis factor-alpha and interleukin-6.

Stimulation of human peripheral blood monocytes with the thyroid hormones tri-iodothyronine (T3) and thyroxine (T4) enhanced their ability to mature into cytologically and functionally characteristic veiled/dendritic cells. Veiled/dendritic cell transition induced by T3 and T4 was dependent on the production of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF alpha) and interleukin-6 (IL-6) in the culture, since the addition of antibodies specific for GM-CSF, TNF alpha and IL-6 to the culture system had blocking effects. The addition of antibodies to macrophage colony-stimulating factor and IL-1 had no effects. Contaminating T cells and B cells did not contribute to the transition of monocytes to veiled/dendritic cells, and it is therefore likely that the GM-CSF, TNF alpha and IL-6 produced in the culture system were derived from the monocytes themselves. Stimulation of the blood monocytes with an optimal concentration of metrizamide (14.5%), reverse T3 (rT3; 2 x 10(-10) M) or highly iodinated thyroglobulin (Tg; 2 x 10(-11) M) also resulted in an increased transition of monocytes to veiled/dendritic cells, but to a lesser extent in comparison with the thyroid hormones (T3, 31 +/- 6% and T4, 25 +/- 5% vs rT3, 22 +/- 8% and Tg with an iodination grade of 0.37%: 20 +/- 4% veiled/dendritic cells). Administration of anti-GM-CSF, anti-TNF alpha and anti-IL-6 to the culture system also had blocking effects on the transition from monocytes to veiled/dendritic cells induced by the iodinated compounds. The mechanisms by which such iodinated compounds act on the monocyte to veiled/dendritic cell transition can only be speculated on (interference H2O2-generating system?).

Dysfunction of monocytes and dendritic cells in patients with premature ovarian failure.

PROBLEM: Due to the presence of ovarian antibodies it has been suggested that premature ovarian failure (POF) belongs to the autoimmune endocrinopathies. Monocytes and the monocyte-derived dendritic cells play a prominent role in the initial stages of endocrine autoimmune reactions: the accumulation of monocytes/dendritic cells and the clustering of dendritic cells in endocrine organs is one of the first phenomena of an autoimmune endocrinopathy. METHOD: This report describes a study on (1) the chemotactic responsiveness of blood monocytes, and (2) the cluster capability of blood dendritic cells in POF patients. The monocyte chemotaxis was determined using the cell's capability to polarize (changes in shape determined by light microscopy) under the influence of the chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (fMLP). The cluster capability of dendritic cells was tested by allowing the dendritic cells to form aggregates with allogenic lymphocytes in vitro. RESULTS: The blood monocytes of 46% of a total of 28 POF patients showed a decreased fMLP induced monocyte polarization in comparison to healthy control values. None of the young female controls (N = 28) and postmenopausal women (N = 17), showed such a defective monocyte polarization. The blood dendritic cells of 36% of the POF patients showed a decreased cluster capability. Defects in monocyte polarization and dendritic cell clustering were not affected by therapies aimed at changes in the estrogen levels or gonadotropin levels of the patients [using estrogen substitution therapy, gonadotropin-releasing hormone (GnRH) analog, follicle-stimulating hormone (FSH)]. CONCLUSIONS: A redistribution of active monocytes and of active dendritic cells from the peripheral blood to the ovaries may be the cause of the described abnormalities. Since similar abnormalities in monocyte function and dendritic cell function have been described in Graves' disease and type I diabetes, the data strengthen the view that POF is one of the endocrine autoimmune diseases.

Defects in monocyte polarization and dendritic cell clustering in patients with Graves' disease. A putative role for a non-specific immunoregulatory factor related to retroviral p15E.

A depressed chemotactic responsiveness of monocytes and a depressed cluster capability of dendritic cells have been found in diseases such as chronic purulent infections of the respiratory tract and in various types of malignancies. These impairments in monocyte and dendritic cell function could be ascribed to the action of a low molecular weight factor (LMWF; less than 25 kDa) circulating in the serum of the patients. The factor, which seems to be a non-specific immunoregulatory factor, shares a structural homology with p15E, the capsular protein of murine and feline leukaemogenic retroviruses. In order to study the chemotactic responsiveness of monocytes and the cluster capability of dendritic cells of Graves' patients, monocytes were isolated from the peripheral blood and dendritic cells were prepared from these peripheral blood monocytes by exposure to metrizamide. Monocytes were studied for their chemotactic responsiveness measuring their capability to polarize (morphological changes determined by light microscopy) after stimulation with the chemoattractant fMLP. Dendritic cells were studied for their capability to form clusters with allogeneic lymphocytes. A defective fMLP-induced monocyte polarization was found (16 vs 37% in healthy controls), whereas the dendritic cells showed a defective clustering (60 clusters vs 151 clusters in healthy controls). The effect of fractions of less than 25 kDa prepared from the serum of Graves' patients on healthy donor monocytes and dendritic cells was studied to test the presence of p15E-like factors. The serum fractions had a significant inhibitory effect on monocyte polarization and dendritic cell clustering.(ABSTRACT TRUNCATED AT 250 WORDS)

A diminished adherence of blood lymphocytes of patients with thyroid autoimmune disease to high endothelial venules in the thyroid and the thyroid-draining lymph nodes.

The function of high endothelial venules (HEVs), present in the T-cell area of lymphoid tissue is to attract lymphocytes to secondary lymphoid organs ("homing"). In Graves' disease, sporadic goitre and lymphocytic thyroiditis HEVs develop in the thyroid. To study the "homing" of peripheral blood lymphocytes (PBL) of healthy individuals and thyroid patients to the thyroid area we studied the adherence of PBL of such individuals to HEVs present in Hashimoto's goitres and to HEVs in thyroid draining lymph nodes. A modification of the in vitro "homing assay" described by Stamper and Woodruff (J Exp Med 144: 823) was used. The number of PBL of patients with Graves' disease which adhered to thyroidal and thyroid-draining lymphnode HEVs was significantly (p less than or equal to 0.001, Wilcoxon test) less than that of healthy control PBL's; in the case of thyroid HEVs 12 (mean, sd 8, n = 18) patient lymphocytes adhered to 35 HEVs vs 19 (mean, sd 7, n = 16) healthy control lymphocytes; in the case of thyroid lymphnode HEVs 20 (mean, sd 12, n = 15) patient lymphocytes adhered vs 35 (mean, sd 9, n = 9) healthy control lymphocytes. PBL of a few sporadic goitre (n = 5) and atrophic lymphocytic thyroiditis (n = 2) patients also showed a diminished adherence to thyroidal HEVs. We also studied the homing capability of lymphocyte suspensions isolated from the thyroid glands of three Graves' disease patients; these infiltrated cells showed a normal adherence pattern to thyroidal HEVs. We favour the idea that the data should be explained by a redistribution of lymphocytes possessing "thyroid-specific-homing-receptors" from the circulation to the thyroid area in patients with thyroid autoimmune disease.

Accessory cells with a morphology and marker pattern of dendritic cells can be obtained from elutriator-purified blood monocyte fractions. An enhancing effect of metrizamide in this differentiation.

Human monocytic cell fractions obtained by counterflow elutriation centrifugation (with regard to specific monocyte/macrophage characteristics: 82-88% were positive for nonspecific esterase; 86-94% for CD14) were cultured (overnight, 37 degrees C) under nonadhering conditions (polypropylene tubes). Thirty to 40% of the cells were found to differentiate into large, monocytoid cells with a dendritic morphology. These cells expressed a marker of active dendritic cells RFD1 in 76-89% and were also positive for class II MHC antigens as identified by OKIa (95-97%). An exposure of the blood monocytic cells to metrizamide (30 min, 14.5%) prior to the overnight culture enhanced this differentiation, and 47-58% of cells with a dendritic morphology were found (of which 80-87% RFD1 positive, and 95-97% Class II MHC positive). The cultured cell populations containing the cells with the morphology and marker pattern typifying dendritic cells, appeared functionally more active as accessory populations when compared to freshly isolated blood monocytic cell fractions; the cultured cells had an enhanced stimulator capability in MLR, and cluster formation with lymphocytes was more prominent. At the same time, the cultured cell population showed a decreased bactericidal activity when compared to the freshly isolated monocytic populations, and in addition, all the cultured monocytoid cells had lost non-specific-esterase activity, while only approximately 10% of cells were still positive for the CD14 marker. When U937 cells were exposed to metrizamide (14.5% concentration, 30 min) and cultured under nonadhering conditions for 36 hours, similar changes were observed (30-45% became dendritic, 20% RFD1 positive).(ABSTRACT TRUNCATED AT 250 WORDS)

High endothelial venules present in lymphoid cell accumulations in thyroids affected by autoimmune disease: a study in men and BB rats of functional activity and development.

High endothelial venules (HEVs) derive their name from the cuboidal high walled shape of the endothelial cells and are found in T-cell areas of a wide variety of lymphoid tissues. The function of HEVs is to attract lymphocytes to lymphoid tissues, and they are thus of importance in lymphocyte recirculation. We investigated the immunohistochemical presence and localization of HEVs in thyroid tissue obtained at surgery from 13 patients with Graves' disease and 3 patients with Hashimoto's disease using the monoclonal antibody HECA 452, which reacts with an epitope on human HEVs. HEVs were found in large confined lymphocytic accumulations in 7 of the 13 Graves' disease glands and all of the Hashimoto's disease glands. These lymphocytic accumulations showed a high grade of architecture and were composed of a central B-cell follicle and a T-cell area in which the HEVs were present. The HEV-containing T-cell area formed a corona around the B-cell follicle. Plasma cells were found at the periphery of the intrathyroidally developed lymphoid tissue. In BB rats, an animal model for spontaneous autoimmune thyroid disease, the development of such HEVs containing intrathyroidal lymphoid tissue was a rather late phenomenon in the autoimmune process occurring after the appearance of anticolloid antibodies in the circulation and after initial immune activity of the thyroid draining lymph nodes. The actual development of the intrathyroidal lymphoid tissue was initiated by accumulation of lymphocytes around dendritic cells forming small cellular clusters. These small clusters later developed into larger formation in which HEVs were detectable. The adherence of B- and T-lymphocytes to human intrathyroidal HEVs was additionally studied using an in vitro adherence assay. B-Lymphocytes preferentially adhered to thyroidal HEVs. This adherence pattern is similar to that of HEVs in mucosa-associated lymphoid tissue (tonsils and Peyer's patches). We conclude that lymphoid tissue with a lymphocyte recirculation pattern similar to that of mucosa-associated lymphoid tissue can be found in thyroid glands involved in a process of autoimmune reactivity; the BB rat study suggests that the development of such tissue occurs during the chronic phases of the process.

Abnormal monocyte chemotaxis in patients with chronic purulent rhinosinusitis: an effect of retroviral p15E-related factors in serum.

Earlier we reported that about 60% of patients suffering from unexplained relapsing of chronic purulent rhinosinusitis show a defective T cell-mediated immunity to commensal microorganisms of the upper respiratory tract. The monocyte chemotactic responsiveness was assessed in 40 of these patients by means of the polarization assay. Impaired FMLP-induced monocyte polarization was found in 26 of the 40 patients tested. The defective chemotactic responsiveness could be explained by a p15E-related factor detectable in the serum of the patients: addition of serum fractions less than 25 kD to healthy donor monocytes resulted in an inhibition of polarization; a monoclonal antibody directed against p15E neutralized this inhibitory effect. In individual patients, a decreased monocyte polarization correlated well with the presence of this p15E-related factor in serum, as well as with defective T cell reactivity.

Immunohistochemical detection of retroviral-P15E-related material in carcinomas of the head and neck.

As has been reported previously, head and neck carcinomas produce low molecular weight factors (H/N ca LMWFs); a molecular weight less than 25000 daltons is capable of inhibiting the chemotaxis of mononuclear phagocytes. The effect of the factors could be neutralized by antibodies to P15E, one of the structural envelope proteins of Murine Leukemia Viruses (MuLV). This indicates that these low molecular weight factors derived from the tumors are related to P15E. In this study, 35 biopsy specimens of head and neck carcinomas were subjected to an indirect immunoperoxidase assay, in order for P15E-like material to be detected morphologically. All head and neck carcinomas gave positive results. Sixty-three percent of other carcinomas (used as controls) were positive as well. P15E-like material was also expressed in epithelia-overlaying inflammatory responses. Healthy epithelia were not positive. This report thus supports the view that P15E-like molecules can be easily detected in cancerous disease--not only by way of biologic isolation, but also by use of immunohistochemical techniques. Since the factor is not specific for the malignant state, it cannot be used as a tumor marker. Possibly involved in the pathogenesis of cancerous disease, its relationship to growth factors, oncogenes, and the immune system needs further clarification.

Immunosuppressive retroviral P15E-related factors in head and neck carcinomas.

We report on the effect of low molecular weight factors (LMWFs) derived from 14 different head and neck carcinomas on the chemotactic responsiveness of healthy donor monocytes, as measured by their polarization activity. The factors inhibited the polarization of the monocytes significantly (61.5% to 94.5% vs 12.5% to 29% in cases where the LMWF was derived from healthy oral mucosa). The inhibitory effect exerted by these LMWFs could be neutralized only by absorption with one of three different murine monoclonal antibodies or a rabbit polyclonal antibody to the murine retroviral envelope protein P15E. This shows that retroviral-related material is present in head and neck carcinomas and that this material is responsible for the observed defective polarization that probably underlies the earlier described defects on monocyte chemotactic responsiveness.

Defective monocyte chemotaxis in patients with head and neck cancer. Restoration after treatment.

Monocyte chemotactic responsiveness (MCR), as measured by the monocytes' capacity to migrate through polymer (Millipore) membranes toward the chemoattractant casein, is impaired in all patients with head and neck cancer thus far examined. Using a more rapid and sensitive test system, the polarization assay, we tested the MCR in 24 patients with head and neck cancer and 31 controls and compared it with the outcomes of the well-established Boyden chamber method. The results of both methods correlated well. All patients showed a seriously depressed monocyte chemotaxis before treatment when tested in the polarization assay. Nine patients were reexamined after surgery, and in seven patients the defective MCR was restored. This illustrates that tumor-derived factors are probably responsible for the inhibitory effect on monocyte chemotaxis. The polarization assay has the potential for predicting early relapse.

Mononuclear phagocyte function in head and neck cancer: depression of murine macrophage accumulation by low molecular weight factors derived from head and neck carcinomas.

In earlier experiments chemotactic responsiveness of peripheral blood monocytes obtained from patients with head and neck cancers was found to be markedly depressed. In an attempt to attribute this defect in migration to an influence excited by low molecular weight factors of less than 25,000 daltons, derived from the tumor, Amicon filtrates of head and neck cancer cells were administered subcutaneously to C3H mice 24 hrs. before the intraperitoneal injection of concanavalin A. Subsequent macrophage accumulation into the peritoneal cavity was quantified. A clear inhibition of macrophage infiltration was found, particularly when filtrates of poorly differentiated tumors were used. Injection of filtrates from healthy oral mucosa were negative, whereas mouse mammary carcinoma filtrates strongly inhibited accumulation.