G. Semenzato

The lymphoproliferative disease of granular lymphocytes. A heterogeneous disorder ranging from indolent to aggressive conditions

A multiparameter analysis, which included the evaluation of clinical features, cell morphology, karyo‐type, phenotypic and functional immunologic findings, and T‐cell receptor beta‐chain configuration was performed on 34 patients with lymphoproliferative disease of granular lymphocytes (LDGL). The two‐fold aim of the study was to identify the most useful tools that would more accurately characterize these patients and to deal with the problem of classifying these lymphoproliferative disorders. The data presented in this article suggest that a single parameter may not be sufficient to define the nature of the proliferating cells or to predict the clinical course of the disease and prognosis for the patient. The use of a multiparameter approach, however, may reach this goal, thus providing important prognostic and therapeutic information. Our study supports the concept that lymphoproliferative disease of granular lymphocytes is a heterogeneous disorder that ranges from indolent and possibly reactive conditions to the manifestation of aggressive malignancies.

Increased Serum Levels of Soluble Interleukin-2 Receptor in Patients with Systemic Lupus Erythematosus and Rheumatoid Arthritis

In this study we investigated the serum levels of a released soluble form of the interleukin-2 receptor (sIL-2R) in 42 patients with rheumatoid arthritis and in 12 cases of systemic lupus erythematosus. Data were evaluated in relationship to the clinical phase and compared with those observed in normal controls (N=56) and in osteoarthritis (N = 7). Increased levels were observed in both rheumatoid arthritis (mean ± SE, 604±49 U/ml) and systemic lupus erythematosus (1438±481 U/ml). These values were significantly higher than in control (256±15 U/ml;P<0.001) and in osteoarthritis (298±33 U/ml;P<0.001) groups. In addition, the highest values were associated with the active phases of both rheumatoid arthritis (active vs inactive, 771±78 vs 451±39 U/ml;P<0.001) and systemic lupus erythematosus (active vs inactive, 2108±489 vs 499±75 U/ml;P<0.001). Our findings suggest that the detection of sIL-2R in rheumatoid arthritis and in systemic lupus erythematosus may represent a good marker of disease activity, which indirectly indicates the ongoing activation and/or proliferation of immunoreactive cells which are involved in the pathogenetic events of these autoimmune conditions.

Immunohistological study in sarcoidosis: Evaluation at different sites of disease activity

Sarcoidosis is a multisystem disease characterized by enhanced immune responses at sites of involvement. For this reason, an immunohistological study using monoclonal antibodies against T-cell subpopulations was carried out in order to evaluate the topographic distribution of immunocompetent cells in tissue sections obtained from a variety of involved organs, such as parenchymal lung, lymph nodes, eyes, skin, and liver. Biopsy specimens were also stained for detection of immunoglobulins, complement, and fibrinogen deposits. The data demonstrate a redistribution of T cells from the blood to all the sites of disease activity where they account for the large majority of infiltrating cells, both in the early lesions (merely a lymphocytic infiltrate) and in well-organized granulomas. Moreover, these cells express a helper-related phenotype, as demonstrated by the high Leu-3/Leu-2 ratios, at sites of involvement with respect to the blood (blood, 1.8/1; transbronchial lung biopsies, 10.5/1; lymph nodes, 19/1; skin, 28/1; liver, 22/1; eye, 14/1). In line with this helper infiltration is the presence of plasma cells and immunoglobulin deposits, suggesting a local hyperreactivity of the B-cell immune system. Both the hypergammaglobulinemia and the T lymphopenia usually observed in the blood of sarcoid patients could be explained by these observations. Comparative analysis of immunohistological data and bronchoalveolar lavage (BAL) findings provides further evidence that BAL cellularity reflects the changes already occurring in lung histology. The studies emphasize the importance that immunological phenomena play in the pathogenesis of sarcoidosis and provide new insights into the mechanisms leading to the formation and maintenance of the sarcoid granuloma.

T lymphocytes in B-cell chronic lymphocytic leukemia: Characterization by monoclonal antibodies and correlation with Fc receptors

In 35 patients with B-cell chronic lymphocytic leukemia (B-CLL), T lymphocytes were characterized by their ability to react with OKT-4 and OKT-8 monoclonal antibodies. These T-cell subsets were also compared with the expression of Fc receptors. An imbalance in the distribution of OKT-4 and OKT-8 lymphocyte subpopulations was observed, with an overall significant reduction in the OKT-4/OKT-8 ratio. When the patients were subdivided according to Rais staging classification, the OKT-4/OKT-8 ratio was more severely impaired in stages III and IV than in stages 0, I, and II. The correlation between the expression of Fc receptors and monoclonal antibodies revealed in both systems an increase in cells bearing suppressor phenotypes (OKT-8+ and TG+ cells) and a decrease in cells bearing helper phenotypes (OKT-4+ and TM+). However, a strict correlation between cells defined by the two assays could not be found in individual cases. In some cases a proportion of T lymphocytes (E+ and OKT-3+) did not express the OKT-4 and OKT-8 determinants; possible implications of this finding are discussed. These data provide further evidence of the T-cell abnormality in B-CLL and emphasize the importance of T-cell subsets in this disease.

T-lymphocyte subpopulations in chronic lymphocytic leukemia: A quantitative and functional study

In the peripheral blood of patients with chronic B‐cell lymphocytic leukemia (B‐CLL) absolute numbers of E‐rosetting lymphocytes were increased. The proportions of TG and TM cell subsets were analyzed, as were their effects on the pokeweed mitogen (PWM)‐dependent differentiation of normal allogenic B cells or of autologous leukemic cells. The TG lymphocyte subset was further studied for its cytotoxic activity in antibody‐dependent cellular cytotoxicity (ADCC). A marked increase both in percentages and in absolute numbers of TG cells was found. TM lymphocytes percentages were normal, but because of the T lymphocytosis occurring in all patients, the absolute numbers of TM were increased. TM and TG subsets showed helper and suppressor activity, respectively, in PWM‐induced B‐cell differentiation. TG cells displayed effector cell activity in ADCC. The results provide further evidence that T lymphocytes from patients with B‐CLL are functionally normal. However, a noticeable increase of the T‐cell subset having suppressor and cytotoxic activity in ADCC was observed. This may be the consequence of a normal immune reaction to the leukemic population.

Activated T cells with immunoregulatory functions at different sites of involvement in sarcoidosis. Phenotypic and functional evaluations

Cells from recovered BAL fluid and from infiltrates in different involved tissues (lungs, lymph nodes, conjunctiva, liver, spleen, and skin) were studied in 22 patients with active sarcoidosis in order to define the surface phenotype, functional in vitro properties, and topographic distribution of the cells in granulomatous lesions. Our data demonstrated a compartmentalization of activated T cells with immunoregulatory functions from the blood to all sites of disease activity. In fact, these cells were found to express the T4+ Leu 8- 5/9+ T17- phenotype, which belongs to cells with helper activity, and that provide heightened responses in functional assays of helper activity, IL-2 release, and the ability to respond in AMLRs. Both a cellular redistribution and a local in vitro replication account for this tissue compartmentalization in sarcoidosis. The microanatomic location of activated T cells, as defined by immunohistological evaluation, showed that the state of activation in these T cells may be a consequence of an intimate contact between helper cells and macrophages within the sarcoidosis granulomas.

Pulmonary alveolar macrophages in patients with sarcoidosis and hypersensitivity pneumonitis: Characterization by monoclonal antibodies

Using a panel of monoclonal antibodies (MoAbs), the frequency of cells bearing Class I and Class II major histocompatibility complex (MHC) determinants, transferrin receptor (TR) sites, and interleukin-2 receptors (IL-2R) has been evaluated on pulmonary alveolar macrophages (PAM) recovered from the bronchoalveolar lavage (BAL) fluid of 21 patients with pulmonary sarcoidosis (including 11 cases with active sarcoidosis and 10 cases with inactive disease), 8 patients with hypersensitivity pneumonitis (HP), and 6 normal non-smoking volunteers. When the frequency of Class II DR-positive cells was considered, 64.3% of control PAM expressed HLA-DR products. No statistically significant differences were observed between controls and sarcoid patients, while HP patients showed an enhanced proportion of DR+ PAM with respect to normal PAM (P<0.05). On the contrary, the frequency of PAM expressing HLA-DQ molecules was higher in both active sarcoidosis and HP patients with respect to patients with inactive sarcoidosis and normal subjects (P<0.001). A statistically significant increase in Class I antigen-positive PAM has been demonstrated in HP patients as compared to controls (P<0.05). Active sarcoid patients showed a higher number of PAM-bearing TR sites than controls and other groups of patients considered (P<0.001). An increase in the percentage of IL-2R-positive PAM has been demonstrated in active sarcoidosis (P<0.001). Our data suggest that (1) PAM of patients with the above-considered interstitial lung diseases are in a state of activation and exhibit structures which play a crucial role in antigenic recognition by T lymphocytes, such as HLA-DQ molecules; (2) the presence of TR in PAM of patients with active sarcoidosis could be related to a more advanced differentiation stage of these cells and/or to particular functional properties; and (3) a direct role of the IL-2/IL-2R system in the interaction between T cells and monocytes in sarcoid lung is crucial. Besides representing an additional parameter which differentiates BAL features of sarcoidosis from those of HP patients, these results could represent a useful tool in the evaluation of the macrophagic component of alveolitis by the BAL.

Immunoregulation in sarcoidosis.

Abstract The immunoregulatory function of lymphocyte subpopulations was studied in 38 sarcoidosis patients subdivided according to their clinical stage and disease phase. Functions studied included the percentage and absolute numbers of TG and TM lymphocytes and their ability to help and suppress the PWM-induced B-cell differentiation of normal allogenic and autologous B cells. The percentage of TG cells was increased in both active and inactive sarcoidosis, whereas their absolute number was increased only in the active phase of disease. This increase in TG cells was unaffected by pronase treatment and the Fc-IgG receptors were modulated as well as normal TG lymphocytes. The percentage of TM cells appeared statistically reduced only in active sarcoidosis while their absolute number, considering the lymphopenia, appears uniformly reduced in both phases of the disease. Functional analysis in the PWM-induced plasma cell generation assay revealed that TG sarcoid lymphocytes retain the property of suppressing B-cell differentiation, suggesting a quantitative imbalance without qualitative alterations. Aside from being diminished, the TM cells were not able to provide an optimal intracytoplasmic Ig production thereby supporting the presence of a qualitative deficiency. B sarcoid cells when cocultured with autologous Tnon-G populations in the presence of a polyclonal activator showed an impairment in plasma cell production; however, B-cell function was almost completely restored when the help was provided by normal Tnon-G lymphocytes, suggesting that the abnormalities observed in sarcoid patients are entirely related to alterations in the T immunoregulatory functions. The implications of the finding of increased suppressor and diminished helper activity in sarcoidosis patients are discussed.

Phenotypical and functional analysis of natural killer cells in sarcoidosis

The frequency of cells reactive with natural killer (NK)-related monoclonal antibodies (MoAbs) HNK-1, NKP-15, B73.1, VEP-13, Ab8.28 has been evaluated in the peripheral blood and bronchoalveolar lavage (BAL) fluid of 39 patients with pulmonary sarcoidosis (including 19 cases with active sarcoidosis and 20 cases with inactive disease). This phenotypic analysis was carried out together with the NK in vitro functional evaluation of cell populations from peripheral blood and BAL fluid. In addition, inhibition studies were performed in order to evaluate the ability of alveolar macrophages (M phi) to modulate NK activity. Data from peripheral blood showed an increased number of mononuclear cells bearing HNK-1, NKP-15, Ab8.28, VEP-13, and B73.1 determinants in patients with active sarcoidosis with respect to patients with inactive disease and controls. The majority of HNK-1-positive cells lacked both Leu2 and Leu3 antigens when investigated in a double marker system. A parallel increase in the in vitro cytotoxicity assay has been demonstrated. On the other hand, only a few mononuclear cells recovered from BAL fluid displayed a surface pattern of NK cells. This small population of HNK-1-positive cells expresses the HNK-1/Leu3 phenotype and does not exhibit NK activity. The alveolar M phi from sarcoid patients, as well as alveolar M phi from controls, have the property of inhibiting the NK activity of autologous peripheral blood lymphocytes. The lack of lung NK function in patients with active sarcoidosis may be related to the presence of immature forms of NK cells and/or to the release of soluble factors by alveolar macrophages.

Lysis of pulmonary fibroblasts by lymphokine (IL‐2)‐activated killer cells—a mechanism affecting the human lung microenvironment?

In this study we investigated whether IL‐2‐activated killer cells may bind and exert lytic activity against non‐transformed lung fibroblasts. We demonstrated that human lymphokine‐activated killer (LAK) cells generated in vitro following incubation with recombinant IL‐2 of either peripheral blood mononuclear cells (PB‐LAK) or lymphocytes obtained from bronchoalveolar lavage (BAL‐LAK), but not resting cells, can lyse normal lung fibroblasts obtained from transbronchial lung biopsies in a 4‐h 51Cr release assay. Both autologous and allogeneic fibroblasts were consistently lysed by LAK cells, thus suggesting that the phenomenon we observed is not MHC‐restricted. Since fibroblasts can bind IL‐2 through specific receptors, we evaluated whether long‐term culture with rIL‐2 could modulate the susceptibility to lysis of target cells. Our data showed that autologous fibroblasts were more resistant to lysis than allogeneic fibroblasts when they were cultured with rIL‐2. Since LAK cells have been demonstrated to release a series of different immunomodulatory cytokines, we evaluated the effect of short‐term incubation of fibroblasts with different factors, including IL‐1, IL‐2, IL‐3, IL‐4, IL‐6, tumour necrosis factor‐alpha (TNF‐α), and interferon‐gamma (IFN‐γ), on the binding and the lysis mediated by LAK cells. These cytokines were not directly cytotoxic on fibroblasts. Only IFN‐γ was found to have a significant protective effect against the lysis. Our data support the concept that a self‐directed cytotoxicity against pulmonary fibroblasts is generated during lymphocyte activation with rIL‐2.