G. Cordier

Activated lung lymphocytes in hypersensitivity pneumonitis

T-lymphocyte activation was investigated in peripheral blood and bronchoalveolar lavage (BAL) of four patients with hypersensitivity pneumonitis. The study was performed by flow cytometry with the use of immunofluorescence labeling with monoclonal antibodies to lymphocyte differentiation or activation antigens. Simultaneous measurement of DNA and RNA content by acridine orange staining was used for cell-cycle analysis. The various cell types were identified by their light-scattering properties. T cell activation was demonstrated in the BAL of all patients by the presence of T cells (OKT3 positive) bearing class II histocompatibility antigen (HLA-DR) and activated T cell markers (MLR 1-3). Lymphocyte proliferation was evidenced in BAL but not in blood of patients by an increased percentage of cells in S + (G2 + M) phases. In addition, T-lymphocyte subsets analysis revealed no abnormalities in the blood and no major imbalance in the BAL despite a slightly increased OKT4/OKT8 ratio. The finding of T cell activation and lymphocyte proliferation in hypersensitivity pneumonitis alveolitis is consistent with the contribution of a local type IV immune reaction to the pathogenesis of this disease.

Anti-CD4 monoclonal antibody administration in renal transplanted patients

Administration of anti-CD4 antibodies in rodents was shown to prevent or to reverse spontaneous or experimentally induced autoimmune diseases and to delay organ or skin allograft rejection. Some anti-human CD4 antibodies were shown to be immunosuppressive when injected in monkeys. BL4, and IgG2a anti-human CD4 murine monoclonal antibody, which binds to an epitope located between the two N-terminal domains of the CD4 molecule, was administered to 12 recipients of a renal cadaver allograft, in association with azathioprine (2.5 mg/kg/day) and prednisolone (1 mg/kg/day). Treatment was started 1 day after transplantation and was discontinued after 3 to 14 days (median 5 days). Infusion of 10 or 15 mg of BL4 over 1 hr induced a selective but transient CD4+ lymphocytopenia. The lack of clinical side effect was remarkable. Acute rejection occurred in 4 out of 12 treated patients. Antibody response to BL4 3 weeks after completion of the treatment was demonstrated in only one patient. Residual antibody concentrations in serum, 24 hr after infusion, ranged from 0.1 to 0.5 microgram/ml, that is below the concentration required to achieve 50% inhibition of allogenic mixed lymphocyte reaction in vitro (1-10 micrograms/ml) or to saturate CD4 binding sites (5-10 micrograms/ml). Rapid degradation and dissociation of cell bound BL4 contributed to the failure to achieve high residual serum levels of the antibody.

In vivo activation of alveolar macrophages in ovine lentivirus infection

Sheep infected by visna-maedi virus, a lentivirus related to the human immunodeficiency virus, develop a chronic interstitial lung disease. Since monocyte/macrophages are known to be specifically infected by visna-maedi virus, we investigated the role of macrophages in the appearance of pulmonary lesions in animals with naturally occurring disease. Alveolitis in maedi leads to a doubling in bronchoalveolar lavage total cell counts and of macrophages as compared to normal sheep. A significant increase in the relative percentage of neutrophils was also observed, accompanied by an increased spontaneous release of neutrophil chemotactic activity by alveolar macrophages of diseased animals, suggesting that they may be activated. Macrophage activation is also demonstrated by the observation of a significant (x3) increase of spontaneous fibronectin release by alveolar macrophages from maedi lungs, and furthermore by the high level expression of major histocompatibility complex class II antigens on most of these cells. Thus viral infection, although restricted to a small population of macrophages, is able to modulate extensive activation of macrophages in the lung. Activated macrophages release mediators likely to play a role in the development of the alveolitis and the parenchymal desorganization. These findings may be relevant to our understanding of the mechanisms by which human immunodeficiency virus infection leads to pulmonary disease other than that caused by opportunistic infections.

Antibody-Dependent Cell Cytotoxicity (ADCC)

The effector cell(s) in human antibody‐dependent cell cytotoxicity (ADCC). with antibody‐coated chicken erythrocytcs as targets, was studied by comparison of cell suspensions from various lymphoid organs and by means of various cell fractionation methods. Effector cells (K) were found mostly in peripheral blood, spleen, and bone marrow but not in tonsils, lymph nodes, and thymus. Effector cells bear Fc receptors and can form EA rosettes with the antibody‐coated target cells. About 1.5% peripheral blood lymphocytes can form ‘high‐avidity’ EA rosettes with targets coated at low antiserum concentration. Most of the effector cells belong to this small subset, as shown by experiments of selective depletion. Removal of most monocytes, T cells, or B cells from, or addition of T‐cell‐specific antiserum to, the effector cell suspensions did not affect ADCC. Effector cells in this model of ADCC therefore lack the conventional B‐ or T‐cell markers but at least some of them are likely to bear C3 receptors.

Flow cytometry analysis of T lymphocytes in sarcoidosis

To examine the immunologic alterations in patients with sarcoidosis we characterized the cell cycle phase of T lymphocytes that were collected from peripheral blood and from bronchoalveolar lavage fluid. T-cell DNA and RNA contents were measured at the single cell level using flow cytometry after staining with the metachromatic fluorochrome acridine orange. T-enriched lymphocyte suspensions were obtained from peripheral blood and from bronchoalveolar lavage in 17 patients with histologically-proved sarcoidosis (10 patients, stage I and 7 patients, stage II) and in 4 patients with acute extrinsic hypersensitivity pneumonitis (AEHP). The percentages of cells in the S + (GS + M) phase in the peripheral blood of the patients with sarcoidosis did not differ from those of healthy control subjects. With bronchoalveolar lavage, however, elevated numbers of T cells in the S + (G2 + M) phase were found in the patients with AEHP and in those with stage II sarcoidosis when compared to patients with stage I sarcoidosis. The cellular RNA content of T lymphocytes from the peripheral blood showed a typical bimodal distribution without difference between patients and control subjects. Conversely, T lymphocytes obtained by bronchoalveolar lavage from patients with AEHP and sarcoidosis had a homogeneous low RNA content which differed from that of T lymphocytes from the blood from that of in vitro phytohemagglutinin-stimulated lymphocytes. These findings provide a new approach to the study of the mechanisms of local T-cell activation in sarcoidosis.

Characterization of the lymphocytic alveolitis in visna‐maedi virus‐induced interstitial lung disease of sheep

In order to investigate the contribution of lymphocytes to interstitial lung disease in animals with visna‐maedi infection, we studied in parallel bronchoalveolar cells and lung tissue from slaughterhouse animals (n= 29) and from colostrum‐deprived lambs transtracheally inoculated with field isolates of visna‐maedi virus (n= 9) or saline (n = 6). Lymphocyte subpopulations were identified in bronchoalveolar lavagc by immunofluorescence and flow cylomctry analysis and in lung tissue using indirect immunohistochemistry. In infected animals a lymphocytic alveolitis containing CD4 and CD8 lymphocytes was observed. Pcribronchovascular lymphoid nodules comprise mostly CD4 lymphocytes. Alveolar lymphocytes of both subsets displayed increased expression of MHC class II antigens in animals with naturally occurring maedi but not in experimentally infected ones. A sequential process of lymphocyte attraction and activation is likely to occur in vivo as part of the alveolitis.

Visna-maedi virus-induced expression of interleukin-8 gene in sheep alveolar cells following experimental in vitro and in vivo infection

Visna-maedi virus is a lentivirus which causes inflammatory disorders in sheep, including a chronic interstitial lung disease resembling that observed in human immunodeficiency virus type 1 (HIV 1) infection. In view of our previous demonstration of the production of neutrophil chemotactic activity by alveolar macrophages, and given the lymphocytic and neutrophilic nature of the alveolar cell infiltrate in both naturally and experimentally infected animals, we hypothesized that interleukin-8 (IL8) could be a candidate for at least part of the chemotactic activity we described. In this study, we investigated IL8 mRNA expression following visna-maedi virus infection. Northern analysis of total RNA using an ovine IL8-specific probe demonstrated that the IL8 gene is upregulated in alveolar macrophages as a consequence of in vitro infection and in alveolar cells from experimentally infected animals. Using a semi-quantitative RT-PCR method, we showed that various levels of IL8 mRNA are expressed by alveolar cells from infected animals and that they correlate with the intensity of the lesions. In conclusion, visna-maedi virus is able to induce IL8 mRNA expression in sheep alveolar cells. Results from in vivo infected animals suggest that IL8 could play a role in the early build-up of visna-maedi virus-induced lesions.

Analysis of Human B Cell Membrane Antigens by Means of Monoclonal Antibodies

Abstract Seven cytotoxic monoclonal antibodies against Raji cells were produced by the hybridoma technique. The specificity of these antibodies for leucocyte populations and the cross reactivity with non-lymphoid cells was analysed. In particular, one of the antibodies, BL11, recognizes peripheral blood B lymphocytes and monocytes as well as certain T lymphocytes. It cross-reacts with epidermal Langherans cells and inhibits the stimulating activity in MLC. The possiblly that BL11 recognizes a HLA-DR epitope common to T cells is discussed.

Keratin filament composition of human epidermal spinous and granular cell fractions selected by flow cytometric sorting

The epidermis, composed mainly of keratinocytes, undergoes continuous proliferation and differentiation characterized by a series of morphological and biochemical changes as cells progress from the basal to the outer cornified layer. One approach to the study of keratinocyte differentiation is to separate the keratinocytes at their successive stages of maturation. Previous attempts to isolate the histologically defined keratinocytes of newborn mouse epidermis have mainly involved the action of trypsin or EDTA combined with separation on density gradation [1, 2, 11]. Fractions enriched in various cell types of human epidermis have been obtained either by serial sectioning parallel to the skin surface [5] or sequential release of cells from the tissue [12]. In the present report, we describe the keratin composition of spinous and granular cell fractions selected by flow cytometric sorting. The specific marker used in this study is the KM48 monoclonal antibody. KM 48 (IgM class) was obtained by immunization of Balb/c mice using a sonicated normal human epidermal cell suspension the which has been shown to recognize a keratinocyte membrane antigen localized at desmosomal regions. Quantitative electron microscopy studies using the immunogold technique have revealed that the expression of the KM48 antigen by keratinocytes increases in parallel with cell differentiation; basal cells were negative, spinous and granular cells were, respectively, mildly and strongly KM48 positive. Corneocytes remained unstained [7].

Cytotoxic and proliferative responses of human thoracic duct lymphocytes: effect of thoracic duct drainage in uremic patients.

Abstract In 22 uremic patients treated by thoracic duct drainage prior to renal transplantation, the cytotoxic and proliferative responses of peripheral blood lymphocytes (PBL) and thoracic duct lymphocytes (TDL) were compared. Renal insufficiency did not reduce the proliferative response to phytohemagglutinin (PHA) but resulted in a profound depression of PHA-induced cytotoxicity and antibody-dependent cell cytotoxicity (ADCC). Depletion of recirculating lymphocytes decreased PHA-induced DNA synthesis but did not affect the cytotoxic responses. TDL were found to be less efficient effectors of ADCC than PBL. Differences between in vitro responses of PBL and TDL as well as the selective effects of renal insufficiency and lymphocyte depletion suggest that the pro-liferative and cytotoxic reactions in the three in vitro systems presently studied are supported by different lymphocyte subsets.