Functional alterations of human blood monocytes after exposure to various nickel compounds in vitro: an effect on the production of hydrogen peroxide.

It is generally known that nickel, a metal with distinct carcinogenic properties, can significantly alter the functioning of host defense mechanisms and impair various components of the immune system. In the present study the influence of 3 nickel salts on the production of hydrogen peroxide (H2O2) by human monocytes was examined in in vitro culture. Highly purified, resting and PMA-stimulated normal human monocytes were cultured with subtoxic concentrations of nickel subsulfide nickel sulfate, nickel acetate and manganese chloride. A portion of the cells was cultured with nickel-manganese salt mixture. Following culture cells were tested in an in vitro functional assay for H2O2 production. It has been shown that all nickel salts, used in micromole concentrations, suppressed H2O2 formation both in resting and PMA-stimulated monocytes, while it was not the case when manganese chloride was used for cell cultures. The strongest suppressive effect was manifested by nickel sulfate. The cells subjected to nickel-manganese mixture displayed H2O2 production similar to that of control ones. These results show that nickel salts in micromole concentrations exert a suppressive effect on oxygen-dependent antimicrobial system of human monocytes and manganese prevents this effect.

The effect of nickel compounds on immunophenotype and natural killer cell function of normal human lymphocytes.

In order to elucidate effects of nickel on human lymphocytes in vitro, peripheral blood mononuclear cells from normal donors were initially tested for viability in the presence of increasing concentrations of two selected nickel salts, sparingly-soluble nickel subsulfide (Ni3S2), and promptly-soluble nickel sulfate (NiSO4). After establishing the toxicity profile, the cells were cultured for 24 h with each compound at three nontoxic concentrations, 0.01 mM, 0.02 mM, and 0.04 mM, to determine its effect on lymphocyte immunophenotype and function. Cells were also cultured in the presence of 0.01-0.04 mM magnesium acetate, Mg(CH3COO)2, while still other cell samples were subjected to a mixture of Mg(CH3COO)2 plus either Ni3S2 or NiSO4 at equimolar concentration. Following the culture, the immunophenotype of the cells was determined by indirect immunofluorescence, using monoclonal antibodies to major differentiation antigens of peripheral blood mononuclear cells, and their natural killer activity toward K562 target cells was measured. Both nickel salts were found to exert distinct effects on lymphocyte phenotype. Exposure of cells to Ni3S2 resulted in the decline of CD4 and natural killer cell populations. NiSO4 diminished the abundance of natural killer cells and, to a limited extent, also of CD4 cells. The nickel salts tested suppressed natural cytotoxicity of peripheral blood mononuclear cells, with Ni3S2 acting more strongly than NiSO4. The addition of Mg(CH3COO)2 to a nickel salt during in vitro culture abolished the above inhibitory effects. Nickel and magnesium salts did not affect CD3, CD8, CD20, and CD11a cell populations. The results indicate that nickel salts have deleterious effects on human peripheral blood mononuclear cells in short-term in vitro culture, but the magnitude of these effects varies, depending on the cell subsets.

Phenotypic and functional characterization of human T lymphocytes expressing a gamma/delta T cell antigen receptor.

Most mature T lymphocytes express CD3-associated antigen receptor molecules (TCR) formed by alpha and beta chains. Recently, a minor subset has been identified that express a different CD3-associated TCR composed of gamma and delta chains. The cell subset expressing TCR gamma/delta differs from conventional T cells in a number of phenotypic and functional characteristics. The simultaneous lack of both CD4 and CD8 antigens at the cell surface allows one to greatly enrich for TCR gamma/delta + cells (by monoclonal antibodies, mAbs and complement). Cloning of CD4-8- peripheral blood lymphocytes revealed that they are homogeneously composed of cytolytic cells which, in most instances, lyse tumor target cells. TCR gamma/delta + cells proliferated in response to allogeneic cells in mixed lymphocyte culture (MLC) and MLC-derived TCR gamma/delta + cells specifically lysed PHA-induced blast cells bearing the stimulating alloantigens, thus providing the formal proof that they recognize (allo)antigens. The use of different mAbs specific for TCR gamma/delta molecules allowed us to identify two distinct subsets which bound BB3 and delta-TCS-1 mAbs, respectively. The BB3-reactive molecules in peripheral blood-derived TCR gamma/delta + cells were represented by C gamma 1-encoded disulphide-linked heterodimers, whereas delta-TCS-1 reacted with C gamma 2-encoded non disulphide-linked molecules. Both BB3 and delta-TCS-1 mAb induced activation of cloned cells expressing the corresponding antigenic determinants (as assessed by measurements of intracellular Ca++ and/or lymphokine production or cytolytic activity).(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of immunophenotype of lymphoid cells isolated from malignant pleural effusions.

Lymphoid cells, isolated from malignant pleural effusions and collected from patients bearing primary lung carcinoma, were examined by means of indirect immunofluorescence and a panel of monoclonal antibodies vs several CD antigens. The percentages of CD4+ T lymphocytes were found to be significantly depressed in malignant effusions as compared to inflammatory ones. In relation to histological type of cancer it was especially evident in squamous cell and anaplastic carcinomas (small and large cell), in comparison to adenocarcinomas. Expression of T cell antigen receptor tau/delta (TCR-1) on T lymphocytes, demonstrated by BB3 MoAb (vs V delta 2), was significantly higher in malignant effusions as compared with non-malignant ones. This was not the case when A13 MoAb (equivalent of TCS 1) was used (vs V delta 1). Percentage values of NK cells, monocyte/granulocyte series activated cells and B lymphocytes did not differ significantly in malignant and non-malignant effusions. It is concluded that these are T lymphocyte subpopulations which are apparently distinct in both effusion groups examined.

Tumor infiltrating lymphocytes in HLA+ and HLA- laryngeal cancer--quantitative approach.

In search of factors governing the accumulation of tumor infiltrating lymphocytes (TIL), frozen sections from fresh surgical specimens of laryngeal carcinoma (n = 36) were tested by alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemistry for monomorphic determinants of HLA class I and class II expression on tumor cells and for the distribution of lymphoid cells bearing CD differentiation antigens. Cell subsets were quantitated in two tumor compartments, tumor mass and tumor stroma, by computer-assisted image analysis. In a portion of examined samples lymphoid cell suspension was isolated from cancerous tissues and assessed by flow cytometry. It has been found that T cells, localized mostly in tumor stroma, were predominant cell population in the tumor microenvironment. Their ability to penetrate tumor mass but not tumor stroma, by CD8+ T cells in particular, but also by natural killer (NK) cells, was associated with HLA class I antigen expression on tumor cells. In flow cytometric analysis activated T lymphocytes (CD3+DR+) were abundant in HLA+ tumors as compared to HLA- ones. In 4 year follow up of 20 patients the mortality was higher in HLA- group but the data were not statistically significant. These results show that HLA class I expression on tumor cells favor penetration of cytotoxic lymphoid cells into tumor mass, at least in the laryngeal cancer.

Immunoregulatory effect of T cell subsets on PWM-induced IgG synthesis by blood lymphocytes in lung cancer.

The mechanism of pokeweed mitogen (PWM) dependent decreased IgG production by blood lymphocytes from lung cancer patients was studied in comparison to control patients and blood donors. It has been shown that the depletion of monocytes has some influence on IgG synthesis but is not a decisive factor. Also, quantitative alterations in the CD4 and CD8 lymphocyte subsets do not significantly influence the PWM stimulation index for IgG synthesis. The assessment of T lymphocyte suppressor activity in lung cancer patients was performed by means of a co-culture with blood mononuclear cells, while helper activity was evaluated through co-culture with donor B lymphocytes. It has been found that lung cancer patient T lymphocytes have no increased suppressor activity, however, especially in the CD4 subset, display the decrease of helper function for B lymphocytes in PWM-induced IgG synthesis. The weakened helper function of CD4 lymphocytes may explain the suppression of specific antibody synthesis do novo which is evident in patients with lung cancer.

Cell migration between graft and host--an analysis with monoclonal antibodies after allogeneic rat kidney transplantation.

In order to analyse migration patterns of donor MHC class II cells out of transplanted kidney and accumulation of host cells within the graft, immunomorphological studies were performed using monoclonal antibodies in rat allogeneic kidney transplantation model. To answer the question of how many donor cells migrate out of the renal cortex MRC 0 x 3 monoclonal antibody (MoAb) against LEW MHC class II antigens was used. In the grafts explanted after 4,24 48 and 73 h, a slow reduction of donor class II cells was observed and some areas in cortex showed only very few, if any, donor cells. At the same time, starting from day 2 after transplantation accumulation of donor cells was found in perivascular spaces. Spleen sections stained at 24, 48, 72 and 96 h after transplantation revealed donor cells present in recipient's spleen. They were detected up to day 3 after surgery. Their numbers, however, decreased after day 2. After 2 and 3 days, accumulations of recipient's cells between tubules were detected. It was found that many cells in infiltrations were stained with anti-T lymphocyte MoAb. Expression of class II antigen on rat kidney cells increases significantly from the day 4 after transplantation.

Assessment of immunophenotype of potentially cytotoxic tumor infiltrating cells in laryngeal carcinoma.

Among host lymphoid cells engaged in anti-tumor defence, tumor infiltrating cells (TIC) are apparently the most suitable for this purpose, due to feasibility of direct contact with tumor cells. The aim of the present study was to evaluate distribution of TIC which express phenotype of cytotoxic cells, in tissues of laryngeal carcinoma. Cryostat sections of surgical tumor samples were subjected to sensitive APAAP immunohistochemistry, following reaction with one of the panel of monoclonal antibodies (MoAbs) vs. several CD antigens and assessed semiquantitatively. It has been found that the content and distribution of potentially cytotoxic cells are quite heterogenous and vary from case to case in examined cancer. CD8+ cells and those bearing NK cell phenotype were the most frequently encountered, mainly within tumor mass. The cells belonging to NK cell subsets, detected by GL183 and EB6 MoAbs could be demonstrated in tumor proximity. TCR-1+ (tau/delta) T lymphocytes were quite a few in part cases. On the other hand, only a scarce number of cells among TIC expressed interleukin-2 receptor. It is concluded that in the vicinity of laryngeal cancer there are fairly large numbers of potentially cytotoxic cells, but at low or nil state of activation.

Protein and mRNA expression of CD56/N-CAM on follicular epithelial cells of the human thyroid.

In order to confirm CD56/N-CAM antigen prevalence in the human thyroid and to compare its expression on thyrocytes and NK cells, an expression of CD56/N-CAM antigen was searched for on isolated thyroid follicular cells and NK cells by flow cytometry. In addition, mRNA for CD56 was searched for in RNA isolated from human thyroid samples and few other organs using dot blot hybridization assay to prove the existence of mechanisms for active synthesis of the protein in question. The isolated cells from the follicular epithelium of 22 various pathological thyroid tissue specimens were examined for the expression of CD56/N-CAM in terms of the percentage of positive cells and the mean fluorescence intensity (MFI). Blood lymphocytes were tested in parallel. The total RNA isolated from thyroid and control tissue specimens was subjected to dot-blot hybridization assay using CD56/N-CAM cDNA probe. All thyroid specimens expressed CD56/N-CAM, but the obtained values differed depending on the tissue examined and the CD56 antibody used. There were no significant differences between the non-malignant thyroid cells of various histology, while cells in the carcinoma group had a much lower MFI, especially the median value. CD56 expression on NK cells from the donors' blood had a homogeneous distribution but the mean and median values of FI were almost three times lower than those on the thyroid cells. Dot blot hybridization came out positive with the RNA isolated from the thyroid specimens and also from the RNA isolated from the tonsil and lymph nodes, but came out negative with the RNA isolated from human and rat kidneys. These results strongly suggest that thyroid follicular epithelial cells express both protein and mRNA of CD56/N-CAM, thus being able to synthesis the relevant antigen. The protein expression seems to be affected by the malignant transformation of the thyroid cells. NK cells have apparently lower CD56/NCAM expression than thyroid cells.

The role of monocytes/macrophages in TCR-zeta chain downregulation and apoptosis of T lymphocytes in malignant pleural effusions.

The aim of this study was to assess alterations of zeta chain expression and their relation to apoptosis of T lymphocytes. T lymphocytes were obtained from malignant pleural effusions (MPE) of 15 patients. The work focused on TCR-zeta chain expression, apoptosis of T lymphocytes, and on the content of monocyte/macrophages in effusions. Analysis was performed using three color flow cytometry combining CD3, TCR-zeta and TUNEL reaction. The content of tumor and monocyte/macrophage cells has been determined using CD3, CD14, and cytokeratins, as markers of distinct cell subpopulations. Our findings strongly indicate that decreased zeta chain expression in T cells depends on the content of monocyte/macrophage cells, and that the range of the decrease is inversely proportional to the number of monocytes/macrophages in the effusion. Those having low zeta chain expression were the main subpopulation of T cells undergoing apoptosis. These data suggest that decreased zeta chain expression in T cells in MPE may be related to the abundance of monocyte/macrophages in effusions.

DNA binding proteins in canine sera. A method for removal of nonspecific DNA binding in the Farr assay.

A panel of canine sera, the majority of which were collected from clinically healthy dogs, were investigated for antibodies against double stranded (dsDNA) by the Farr radioimmunoassay technique. Non-specific DNA binding agents interfering with the Farr assay were detected in all sera. Heat inactivation at 60 degrees C or treatment with dextran sulphate was shown to eliminate this kind of unspecific DNA binding while not affecting true antibodies to dsDNA. Canine sera positive in the Farr assay after inactivation at 60 degrees C were positive also in immunofluorescence for anti-nuclear antibody on rat liver sections and for dsDNA with Chrithidia luciliae as antigen preparation. IgG or glycoprotein nature of the non-specific DNA binding could be excluded by means of affinity chromatography on protein A and the lectin lentil.

Significance of cell adhesion molecules, CD56/NCAM in particular, in human tumor growth and spreading.

Cell adhesion molecules (CAM) represent a large group of cell surface protein moieties with distinctive biological functions. In physiological terms they ascertain cell to cell contact such as cell cohesion of epithelia, condition cell migration and transmigration via biological membranes such as blood vessel walls, provide means for homing cells in a new microenvironment etc. These features of CAM are exploited by tumor cells to grow and spread in a tumor bearing host. CD56/N-CAM antigen is 140 kD isoform of neural cell adhesion molecule. N-CAM belongs to the large Ig superfamily of CAMs. CD56 can be traced at various sites, including nervous tissue, neuro-muscular junctions, neuroendocrine and endocrine organs. It is well known as a differentiation antigen of natural killer (NK) cells. Its role and function are far from clear, but its adhesion properties are evident in cell-cell (homophilic) interactions. CD56 has been, however, demonstrated the cells various human malignancies. Tumors of the nervous system such as neuroblastoma, are well known to express this marker. Malignant lymphomas of T-NK cell origin bear CD56, as well as multiple myeloma, melanoma and some cancers of epithelial origin. These data suggest that CD56/N-CAM antigen is, in some unknown manner involved in tumor biology.

Immunofluorescent analysis of antibodies against neurons in the case of paraneoplastic syndrome.

The authors report clinical and neuropathological findings especially immunofluorescent detection of antineuronal antibodies in the case of paraneoplastic syndrome in course of the small-cell lung carcinoma. The clinical symptoms, observed in 48-year-old woman, covered bilateral pyramidal syndrome, cerebellar syndrome, myasthenic syndrome and impairment of the cranial nerves. Neuropathological investigation revealed paraneoplastic encephalopathy in the form of encephalitis. Immunofluorescent analysis showed brightly fluorescent neurons standing out against a dull background.

Expression of CD56/N-CAM antigen and some other adhesion molecules in various human endocrine glands.

CD56/N-CAM antigen, 140 kDa isoform of neural cell adhesion molecule (N-CAM) has been previously traced by some of us in follicular epithelium of human thyroid by immunohistochemistry. The reaction product was cell membrane bound, being stronger in hyperactive thyroid as compared to colloid goiter. In the current study, CD56 was searched in other endocrine glands and their tumors including parathyroids, adrenal cortex and parafollicular C cells of the thyroid (TT cell line). The antigen was also examined in the tissue extracts of endocrine and nonendocrine organs by dot blot immunoassay and anti CD56 monoclonal antibody. Besides, some other cell adhesion molecules (CAMs) were looked for in the tissues and cells tested. It has been found that CD56 is expressed in all zones of adrenal cortex, albeit in various intensity. The reaction was cell membrane bound in cortical hyperplasia and adenoma but cytoplasmic in the carcinoma of adrenal cortex. Other endocrine tissues and cells tested were devoid of CD56. Presence of CD56 antigen could be confirmed by dot blot assay with 3M KCl and NP40 extracts of both, thyroid and adrenal glands. Apart from CD56 some other CAMs could be traced in thyroid cell membranes including CD44, VLA-3 integrin and E-cadherin, what was not the case in the adrenal cortex. In parathyroids and parathyroid adenoma, diffuse immunostaining of E-cadherin and irregular, focal expression of CD44 was observed. These results show, apart from CD56, abundance of other CAMs in the thyroid gland and their relative scarcity in other endocrine tissues tested.

CD56(NCAM) antigen in glandular epithelium of human thyroid: light microscopic and ultrastructural study.

CD56 antigen, an isoform of the neural cell adhesion molecule (NCAM) was previously found by us in human thyroid by APAAP immunohistochemistry in light microscopy on frozen tissue sections. In the current study, it was attempted to trace the antigen in question using another light microscopic immunohistochemical procedure and to validate the results at the ultrastructural level. For light microscopy, cryostat sections of 12 surgical samples of human thyroid were subjected to ABC (preformed avidin-biotin-peroxidase complex) method. For immunoelectron microscopy, immunoperoxidase reaction was carried out on prefixed, small thyroid tissue blocks. Following preliminary inspection of semithin sections, ultrathin sections were examined in the transmission electron microscope. ABC reaction revealed distinct specific CD56 staining of thyrocyte cell membranes. The staining was weak or absent in thyroid papillary carcinoma cells. The results were confirmed in semithin sections by indirect immunoperoxidase. The latter reaction in ultrathin sections at the ultrastructural level has shown that specific reaction product was confined to free and lateral surfaces of thyroid follicular cells. Endothelial cell membranes of thyroid capillary vessels were totally devoid of the reaction product. The reaction was weakly positive in thyroid follicular and papilllary carcinomas but absent from medullary carcinoma.

Incidence of alloantibodies and immune complexes in bronchial secretions of lung cancer patients.

Bronchial secretions may accumulate immunoglobulins (Igs) produced by lymphocytes infiltrating tumor and its vicinity, and tumor associated antigens to higher degree than cancer patient serum. All three classes of Igs were increased in bronchial secretions from lung cancer patients as compared to control patients. Similar relationships were found for IgG and IgA/albumin ratios and IgG and IgA indices. The level of alloantibodies to fetal cells (measured by immunoradiometric--IRMA--assay) and immune complexes (IC, measured by IRMA--Clq and ELISA--anti C3) were significantly higher in lung cancer patients versus control patients in bronchial secretions. The differences were similar in sera but they were not significant. On the contrary, alloantibody binding to tumor cells was higher in control group. Absorption experiments with normal cells abolished antitumor and antifetal activity, what indicates natural character of these alloantibodies. Significant correlations between alloantibody to fetal cells and IC levels indicate that fetal antigens may contribute to antigenic component of IC. These findings suggest the link of alloantibodies and IC levels in bronchial secretions with neoplastic growth.

Identification of a tumor-related protein antigen in immune complexes derived from pleural effusions of patients with bronchial carcinoma.

Pleural effusions from 15 patients with advanced primary bronchial carcinoma, from 2 patients with metastatic lung cancer and from 6 patients with nonmalignant disease were studied. Immune complexes were found in examined fluids in amounts corresponding to 2.5-210 mg/100 ml of aggregated IgG by means of ELISA solid phase anti C3 and 125ICIq binding radioimmunoassay. Following determination of protein content and salting out by ammonium sulfate of examined fluids, the sediments were subjected to subsequent chromatographic procedure including molecular sieving (Sephadex G-200, Sepharose 4B) and affinity chromatography on Protein A-Sepharose CL-4B. The yield--apparently pure immune complexes--was then split by means of chaotropic agent 2.5 M KSCN. It permitted to obtain 2 fractions: one contained IgG while the other was a non-Ig protein of m. w. = 150 000. The latter isolated from malignant effusions possessed antigenic activity in the leukocyte migration inhibition (LMI) assay. It resulted in inhibition of migration of allogenic peripheral blood leukocytes from lung cancer patients in 87% of cases. It had no activity against leukocytes from nonmalignant disease patients. LMI activity of the final second fraction derived from malignant effusion was significantly different from that of other fractions obtained both from malignant and nonmalignant fluids.

Anti-tumor antibodies in lung cancer patients. Immunofluorescence study using various indicator cells.

By means of indirect immunofluorescence a number of primary lung cancer patient sera and control sera were tested for anti-tumor antibody activity on living tumor cells as a substrate. Antibodies against surface antigens were the most frequently detected in autologous system (in 65%) on cells derived from fresh surgical material of lung cancer. They were also found in 50% of cases using tumor cells from primary short-term culture. When established cell line of lung cancer was used (E-14) in allogeneic system, the antibodies were detected in only 22% of examined lung cancer sera. Absorption of positive sera with homogenates of normal tissues did not abolish their specific activity. Positive reactions were confined to squamous cell type of bronchogenic carcinoma.

Immunofluorescent assessment of tumour infiltrating cells in laryngeal carcinoma. Application of monoclonal antibodies.

In order to gain some insight into host cell accumulations within primary tumour, frozen sections from surgical specimens of laryngeal carcinoma were subjected to indirect immunofluorescence using a panel of monoclonal antibodies against various human lymphocyte subsets as well as macrophages. In addition, polyclonal antibodies against Ig were used in order to trace B cells. Numerous host cell infiltrates seen at the tumour periphery were composed of T4 (helper) lymphocytes and macrophages. Lymphocytes of OKT8 (suppressor/cytotoxic) and Leu-7 (NK cells) series were intermingled with tumour cells in the case of scanty infiltrates. Infiltrating cells were also linked to the presence of metastases in regional lymph nodes. OKT4-positive abundant infiltrates were usually accompanied by uninvolved nodes, while scanty ones with OKT8 specificity were relatively frequently seen in the patients with evidence of nodal metastases. These differences were not statistically significant, however, B cells as well as plasma cells were infrequently observed and were encountered both in tumour samples with intensive cellular infiltrates as well as in those with scanty ones.

Evaluation of phenotype of mononuclear host cells isolated from primary tumour and peripheral blood of patients with laryngeal carcinoma.

Mononuclear host cells isolated from primary laryngeal carcinoma were assessed by means of indirect immunofluorescence with a panel of monoclonal antibodies against various lymphocyte subsets and macrophages. Tumours of various staging groups were examined in parallel with cells isolated from patient and donor peripheral blood (PBL). It was found that percentage values of cells bearing T3 and T4 phenotype were reduced both in tumour infiltrating cells (TIC) and in PBL population. The fall in T4+ cells in PBL from cancer patients in T3 and T4 staging groups was statistically significant (p less than 0.01) as compared with donor cells. Corresponding values for T8+ cells from TIC were increased in T1 and T2 staging groups of cancer, but showed a gradual fall in advanced stages. The T4+/T8+ cell ratio was decreased in both TIC and PBL cells. The HNK-1+ (NK) cell pattern in TIC was analogous to that for T8+ cells, i.e. the cell percentage decreased with advance in tumour growth. Corresponding values for OKM-1+ were increased in TIC and in patient blood, though in TIC they grew in proportion to tumour growth. Ia+ (HLA-DR+) cells in peripheral blood were significantly increased in patients versus those of donors (p less than 0.01), but only in T3 and T4 staging groups of examined cancer. These results show that subsets of tumour infiltrating cells in laryngeal carcinoma are a complex phenomenon, associated with growth and progression of tumour.