Leu 3a Positive Cells Research Articles

Evaluation of a filtration lymphocytapheresis (LCP) device for use in the treatment of patients with rheumatoid arthritis. 1991.

Abstract: Guest Editor's Introduction: Cell adhesion on a material is caused by the interaction between them and related to the material characteristics including chemical structure, surface charge, wettability, smoothness, and microstructure such as microdomain structure. In addition, Nishimura of Asahi Medical Co. found that the size of the material is important particularly in the case of lymphocyte adhesion. Based upon this result Asahi Medical Co. developed lymphocyte removal filters by use of ultra fine fibers having a diameter of 1.7 μm. The filter was first used for leukocyte filters for blood transfusion, and then applied for therapeutic use in the treatment of patients with various hematological and autoimmune diseases. This paper describes the specification of the filter and clinical application for the treatment of rheumatoid arthritis. This paper was printed in Artificial Organs, vol. 15, page 180–188 (1991) and reprinted here with permission.A practical on‐line lymphocytapheresis (LCP) system using a leukapheresis filter (Cellsorba, Asahi Medical Co.) was evaluated in six patients with refractory rheumatoid arthritis. This filter consists of nonwoven fine polyester fiber wound around a porous cylinder. The blood was passed through the polyester fiber at a flow rate of 50 ml/min for 60 min. LCP was carried out once a week in the first month and biweekly in the next 2 months. An average of 98% of the leukocytes that entered the filter (1.27 × 1010 cells) and 100% of the lymphocytes that entered the filter (3.66 × 109 cells) were removed in the first LCP. A total of 96.6% of the platelets and 2.7% of the erythrocytes that entered the filter were also removed. All of the patients showed clinical improvement in morning stiffness, Lansbury articular index, and functional capacity, with no adverse reaction. The number of circulating lymphocytes erythrocytes, and platelets and the concentration of various serum components showed no significant change during the treatments. This LCP system required no fresh frozen plasma, albumin, or other blood transfusion. The number of circulating lymphocytes decreased to 65–70% of the pretreatment circulating lymphocyte count at the last procedure, with a decrease in the ratio of Leu3a positive cells to Leu2a positive cells. The proliferative response to phytohemagglutinin and concanavalin A was improved. These data suggest that LCP to remove ∼3 × 109 lymphocytes once a week or biweekly has an immunomodulatory effect.

肺病変を伴ったＳｊｏｅｇｒｅｎ症候群患者の末梢血活性化リンパ球に対するエリスロマイシンの効果

We evaluated the effect of low-dose long-term erythromycin (EM) therapy (600 mg/day) on activated lymphocytes in the peripheral blood of the patient with Sjögren's syndrome accompanied with lung involvement. Before and after EM therapy, we analysed the subsets of peripheral lymphocytes using two-colour analysis by FACScan. Used monoclonal antibodies were Leu 2a, Leu 3a, Leu 4 and Leu HLA-DR. After 9 months of EM therapy, the percentages of activated Leu 2a positive cells, Leu 3a positive cells, and Leu 4 positive cells significantly decreased.These results might be useful to clarify the mechanism of low-dose long-term EM therapy against diffuse panbronchiolitis.

Evaluation of a Filtration Lymphocytapheresis (LCP) Device for Use in the Treatment of Patients with Rheumatoid Arthritis

A practical on-line lymphocytapheresis (LCP) system using a leukapheresis filter (Cellsorba, Asahi Medical Co.) was evaluated in six patients with refractory rheumatoid arthritis. This filter consists of nonwoven fine polyester fiber wound around a porous cylinder. The blood was passed through the polyester fiber at a flow rate of 50 ml/min for 60 min. LCP was carried out once a week in the first month and biweekly in the next 2 months. An average of 98% of the leukocytes that entered the filter (1.27 x 10(10) cells) and 100% of the lymphocytes that entered the filter (3.66 x 10(9) cells) were removed in the first LCP. A total of 96.6% of the platelets and 2.7% of the erythrocytes that entered the filter were also removed. All of the patients showed clinical improvement in morning stiffness, Lansbury articular index, and functional capacity, with no adverse reaction. The number of circulating erythrocytes and platelets and the concentration of various serum components showed no significant change during the treatments. This LCP system required no fresh frozen plasma, albumin, or other blood transfusion. The number of circulating lymphocytes decreased to 65-70% of the pretreatment circulating lymphocyte count at the last procedure, with a decrease in the ratio of Leu3a positive cells to Leu2a positive cells. The proliferative response to phytohemagglutinin and concanavalin A was improved. These data suggest that LCP to remove approximately 3 x 10(9) lymphocytes once a week or biweekly has an immunomodulatory effect.

Immunohistochemical studies on dust mite antigen in positive reaction site of patch test.

We demonstrated that eczematous reactions could be induced by patch testing with mite antigens in the majority of patients with atopic dermatitis (AD). By using immuno-double labelling technique, many mite antigen-bearing Langerhans cells were seen in the epidermis in the early stage of the patch testing. Twenty-four hours later, these cells were observed only in the deep dermis. Immunoelectron microscopically, it was found that the mite antigens were trapped by macrophages, which were apposing lymphocytes. On the other hand, we observed that Langerhans cells and Leu 3a positive cells in the AD lesions carried IgE molecules. Furthermore, many IgE-positive dendritic cells bearing mite antigen were seen in the positive patch testing sites. Taken together, IgE-mediated contact hypersensitivity to mite antigen may play an important role in the pathogenesis of AD.

Two-color analysis of lymphocyte subpopulations in membranous nephropathy and minimal change nephrotic syndrome

Two-color flow cytometry was carried out to determine the correlation between cell mediated immunity and the development of the nephrotic stage in patients with membranous nephropathy (MN) and minimal change nephrotic syndrome (MCNS). In this study, lymphocyte subpopulations were measured by two-color flow cytometry using various monoclonal antibodies of the Leu series. Thirty patients with MN and 25 patients with MCNS were examined. Clinically, those patients were divided into four stages as follows: (1) untreated nephrotic stage, (2) prednisolone (PSL) treated nephrotic stage, (3) persistent proteinuria stage (incomplete remission, ICR), and (4) complete remission (CR). Pathologically, the patients with MN also divided into four stages I-IV, according to Churg's classification. The values of the Leu 3a/Leu 2a ratio in patients in the untreated nephrotic stage of MN and MCNS were significantly higher than those in the remission stage in both diseases (P less than 0.01, P less than 0.05, respectively). Two-color flow cytometry showed that the reduction of Leu2a positive cells was mainly due to a decrease of Leu 2a+15+ subsets (suppressor T cells) in the untreated nephrotic stage and relative increase of Leu 3a+8+ subsets (suppressor inducer T cells). There was no significant difference in these findings among the histopathological stages in patients with MN. Patients with MN and MCNS showed a significant elevation of Leu 2a+DR+ cells after the treatment of PSL. The abnormalities of suppressor T cells and suppressor inducer T cells in the peripheral blood appear to be correlated with clinical activities of the nephrotic syndrome due to MN or MCNS, but not to be related to the pathogenesis of either disease. It is postulated that PSL might stimulate Leu 2a positive cells and Leu 3a positive cells, and then increase the number of Leu 2a+15+ cells in the peripheral blood of patients with MN and MCNS.

IMMUNOHISTOCHEMICAL STUDY OF THE CELLS INFILTRATING HUMAN RENAL ALLOGRAFTS BY THE ABC AND THE IGSS METHOD USING MONOCLONAL ANTIBODIES

Sixty renal allograft tissues obtained from 29 patients were stained with hematoxylin and eosin. These tissues were histologically classified into 4 patterns according to the distribution pattern of the infiltrating cells: normal, focal, focal-diffuse, and diffuse types. Clinical signs of acute rejection were observed in 88% of the patients with the diffuse type infiltration, and 83% of those with the focal-diffuse type infiltration but in only 13% of those with the focal type infiltration. Twenty-four renal allografts were analyzed by the ABC and the IGSS methods using monoclonal antibodies. The number of T cells (Leu 1) accounted for about 80% of the total number of infiltrating cells; 2-8% of the cells were B cells (Leu 12); about 10% were NK/K cells (Leu 7); and 4-6% were monocytes/macrophages (Leu M3). As to helper/inducer T cell (Leu3a) and killer/suppressor T cell (Leu2a), which are T lymphocyte subsets, there were more Leu3a- than Leu2a-positive cells in focal type tissue, but there were more Leu2a- than Leu3a-positive cells in focal-diffuse and diffuse type tissue. In most cases that developed clinical signs of acute rejection, there were more Leu2a- than Leu3a-positive cells. The Leu3a/Leu2a ratio in most of the AZA-administered cases dropped immediately after the transplantation and maintained a low value, but in the CSA-administered cases it decreased gradually post-transplant.

Expression of class II antigens by subsets of activated T cells

Gene products coded for within the HLA complex play an important role in the control of immune responses. Class I antigens, coded for by the HLA-A, B, and C loci, are expressed by virtually all mononuclear blood cells. Class II antigens, coded for by the DR, DQ, and DP loci, have a more limited tissue distribution. They are expressed by B cells, monocytes, and by activated, but not by resting, T cells. The class II molecules of B cells and antigen-presenting cells have long been of interest to immunologists, since they are involved in the presentation of antigen, in communication between T cells and B cells and between T cells and adherent cells, and in susceptibility to certain diseases. The class II antigens expressed by activated T cells, however, remain largely uncharacterized in terms of their specificity, functional significance, and molecular nature. We have studied the expression of DR and DQ antigens by activated T cells and then examined the expression of DR versus DQ antigens by Leu 2a and Leu 3a subsets of mitogen-activated populations. Our results demonstrated that, as for class II-positive macrophages, the intensity of staining with monoclonal antibodies directed against DR antigens was much greater than that obtained with those directed against DQ antigens. Interestingly, the percentages of Leu 2a- and Leu 3a-positive cells which expressed DR antigens were quite similar, as were the percentages of Leu 2a and Leu 3a cells which expressed DQ. Thus, there does not seem to be preferential expression of DR versus DQ antigens by mitogen-activated T-cell subsets. Finally, though both DR-positive-DQ-positive and DR-positive-DQ-negative populations were detected, few or no DR-negative-DQ-positive cells were observed in these populations.

Identification of inflammatory cells infiltrating renal allografts.

Eight nephrectomies and 13 biopsies of renal allografts (living 15, cadaveric 6) were investigated on the origin of inflammatory cells in the graft tissues by the use of an immunohistologic method (ABC method). Various monoclonal antibodies and heterosera were used to identify different leukocyte subsets. In all specimens the most predominant inflammatory cells were T cells. Other cells decreased in the following order; B cells, neutrophils and monocytes, and natural killer cells. In T cell subsets Leu 2a-positive cells (suppressor/cytotoxic T cells) predominated over Leu 3a-positive cells (helper/inducer T cells) in 4 nephrectomies (living 1, cadaveric 3) and 12 biopsies (living 11, cadaveric 1). Among these 16 cases, 7 were undergoing acute rejection in various degrees, and 9 were without clinical rejection. In the former 7 cases only one was suffering from another disease. In contrast, Leu 3a-positive cells predominated over Leu 2a-positive cells in 1 biopsy (living) and 4 nephrectomies (living 2, cadaveric 2). Four of these 5 cases concurrently had other diseases in addition to acute rejection. Two cases underwent acute tubular necrosis (cadaveric graft nephrectomies) and 2 underwent chronic rejection and crescentic glomerulonephritis (living graft nephrectomies). The one remaining case was without clinical rejection.

Analysis of peripheral blood lymphocytes of atomic bomb survivors using monoclonal antibodies.

In order to study the effects of exposure to atomic bomb radiation on the immune competence of man, the proportions of peripheral blood lymphocyte subsets (subpopulations) were determined by an indirect immunofluorescence antibody assay using monoclonal antibodies and fluorescence microscopy. The study was based on a total of 104 Adult Health Study participants in Hiroshima, including 29 individuals exposed to 100+ rad, 46 exposed to 1-99 rad, and 29 0 rad controls. No change in the proportion of Leu-1 positive cells (total T cells) and Leu-2a positive cells (cytotoxic/ suppressor T cells) and the ratio of Leu-3a/Leu-2a was observed with age, while Leu-3a positive cells (helper/ inducer T cells) decreased with age and HLA-DR positive cells (B cells and monocytes) increased with age, with the differences occurring predominantly in the oldest age group (age > 75). The proportion of HLA-DR positive cells was higher in males, but there was no significant sex difference in the proportions of other cell types and the ratio of Leu-3a/Leu-2a. Radiation exposure did not significantly affect the proportions of Leu1, Leu-2a, Leu-3a, and HLA-DR positive cells and the ratio of Leu-3a/Leu-2a. No interaction between the effects of age and radiation exposure was demonstrated.

Inflammatory cell types in normal human epidermis--an immunohistochemical and morphometric study

Inflammatory cells in 15 specimens of normal human epidermis were selectively stained by a monoclonal antibody immunoperoxidase technique. The quantitative assessment using an interactive image analysis system revealed OKT 11 positive cells (T lymphocytes) and Leu 2a positive cells (suppressor/cytotoxic cells). As there was no significant difference in the distribution of these markers, helper/inducer cells obviously are not present in considerable amounts. OKM 5 positive cells outnumbered OKM 1 positive cells, indicating the presence of a OKM 5+, OKM 1-macrophage subset. The epidermal dendritic cells clearly showed a striking heterogeneity regarding the expression of HLA-DR (62% of OKT 6-positive cells) and Leu 3a (47%), suggesting the existence of immunologically distinct subsets of human epidermal dendritic cells.

Production of interleukin 2 (IL 2) by salivary gland lymphocytes in Sjögren's syndrome. Detection of reactive cells by using antibody directed to synthetic peptides of IL 2.

Because abnormalities in interleukin 2 (IL 2) production have been reported in the blood of patients with certain autoimmune diseases, we have examined the lymphocytes from patients with Sjögren's syndrome (SS) in which it is possible to obtain simultaneous samples of inflammatory site (i.e., salivary gland) lymphocytes and blood lymphocytes. We found that IL 2 production by peripheral blood lymphocytes (PBL) after mitogen stimulation was markedly diminished (4 +/- 2 U/ml) in 8/32 SS patients. However, salivary gland lymphocytes (SGL) from six out of six SS patients (including three patients with low IL 2 production by their PBL) had a high level of IL 2 production (97 +/- 32 U/ml), suggesting that IL 2 production by inflammatory site lymphocytes may differ from blood lymphocytes in the same patients. Low IL 2 production by a patient's PBL was not correlated with the patient's age, duration of disease, immunoglobulin level, or presence of antinuclear antibodies. Low IL 2 production was associated with a decreased ratio of Leu-3a/Leu-2a positive cells (p less than 0.05) and with an increased proportion of "activated" T cells expressing HLA-DR and gp140 (p less than 0.05). To determine the proportion of PBL and SGL containing cytoplasmic IL 2-like material, we used affinity-purified rabbit antibodies prepared against chemically synthesized peptides of human IL 2. Before mitogen stimulation, PBL were not stained by these antibodies (less than 1% reactive cells), whereas SGL T cells eluted from the salivary gland of SS patients contained a small (3.4% +/- 1.8) proportion of reactive cells. A similar proportion (2.4% +/- 1.2) of reactive cells was noted when frozen tissue sections of salivary gland biopsies were examined with these antibodies. After mitogen stimulation, 35% +/- 17 of PBL and 56% +/- 18 of SS SGL were specifically stained with anti-IL 2 peptide antibodies. In summary, these studies demonstrate a significant difference in IL 2 production between PBL and SGL of the same patients. Furthermore, antibodies against IL 2 peptides provide a powerful tool for detection of T cells producing IL 2 in vitro and in situ, and for understanding the role of this lymphokine in pathogenesis.

Identification of lymphocyte subsets and natural killer cells in head and neck cancers. An immunohistological study using monoclonal antibodies.

We investigated host-immune defenses against head and neck cancer cells by using various monoclonal antibodies with an immunoperoxidase technique to define lymphocyte subsets and natural killer (NK) cells. By so doing, we were able to identify lymphocyte subsets and NK cells in various head and neck cancers. We found that the majority of these cells infiltrate in or around nests of cancer cells and are stained with Leu-1 antibody. They include both Leu-2a and Leu-3a positive cells, which show equally intense levels of infiltration. Leu-7 positive cells were only scattered in the peripheral portion of the cancer nests in some cases. We also found a tendency for T-cells to infiltrate more intensely in poorly differentiated squamous cell carcinomas (SCC) than in moderately or well-differentiated SCC. Similarly, T-cells were more prevalent in maxillary carcinomas than in laryngeal carcinomas. These findings suggest the presence of a host-immune defense mechanism against cancer cells in patients with head and neck cancers.

Current research on immunopathology of melanoma: analysis of lymphocyte populations in relation to antigen expression and histological features of melanoma

Immunoperoxidase localization of monoclonal antibodies in sections of melanoma has been used to identify histological features which may be of prognostic importance in melanoma, in particular whether certain structures on melanoma cells may determine the degree and nature of lymphoid infiltrates and whether these may be related to prognosis. Monoclonal antibodies to lymphocyte subpopulations were used to identify and quantitate lymphoid infiltrates in 15 primary melanomas and 8 cutaneous metastases. These were correlated with histological features identified in routine sections. There was a wide variation in the numbers of mononuclear cells associated with both primary and metastatic melanoma. T cells and to a lesser extent macrophages accounted for the majority of the cells, B cells and natural killer (NK) cells for only 2% of the infiltrates. The Leu 3a (helper) T cell subpopulation predominated in primary tumours, OKT8 positive (suppressor/cytotoxic) cells in metastases. Infiltrates in which OKT8 cells predominated were associated with ulceration, a high mitotic rate and thick primary tumours. The converse applied to Leu 3a infiltrates. Infiltrates with a high proportion of Leu 3a positive cells tended to be associated with Thy-1 positive, DR antigen negative tumours. Thy-1 antigens were predominantly expressed on primary tumours and rarely on metastases whereas the converse applied to expression of the acute lymphoblastic leukemia antigen (CALLA) on melanoma cells. These findings suggest that certain melanoma antigens may be related to the nature of the lymphoid infiltrate associated with melanoma and possibly with the behaviour of the tumour in the host. They further suggest that identification of cell surface structures and lymphoid infiltrates by these techniques may be a valuable extension of routine histopathological assessment of prognosis in melanoma.

Local and systemic immune response in multiple sclerosis: analysis of CSF inflammatory changes and peripheral blood T-cell subsets.

In order to investigate the significance of the immune response in multiple sclerosis (MS), we have compared changes in peripheral blood T-cell subsets to the cellular and humoral responses in the cerebrospinal fluid. Using monoclonal antibodies defining the Leu-3a and Leu-2a phenotypes, we found a highly significant (2P less than 0.001) increase of the Leu-3a/Leu-2a cell ratio in peripheral blood T-cells of multiple sclerosis patients with disease activity (acute phase or progressive form), while patients with inactive disease did not differ from controls. No characteristic alterations in the CSF inflammatory response could be correlated with changes in T-cell subsets in the peripheral blood. There was no significant difference in the cerebrospinal fluid cell count or the mean IgG index between patients with high or low Leu-3a/Leu-2a cell ratios. We conclude that increased CSF IgG synthesis in MS is probably not a direct consequence of the apparent reduction of Leu-2a positive cells in the peripheral blood. Thus the biological significance of the change in immunoregulatory cells in the blood of multiple sclerosis patients remains unknown.

Lymphocyte subpopulations and T-cell subsets in human oral cancer tissues: immunohistologic analysis by monoclonal antibodies.

A series of T-cell-specific monoclonal antibodies (Leu-1, Leu-2a, and Leu-3a) and B-cell-specific monoclonal antibody (HLB-1) were used to detect the localization and intensity of infiltration of lymphocyte subpopulations and T-cell subsets in frozen sections of 17 patients with the oral cancer. The vast majority of the lymphocyte infiltrates in the oral cancer tissues were reactive with Leu-1. In contrast, B cells were detectable with HLB-1 in only 2 of 17 cases. Leu-2a-positive cells were dominant in four cases, whereas Leu-3a positive cells were dominant in only three cases. In seven cases, both cells infiltrated to the same degree. Leu-2a positive cells tended to be dominant in the cases with earlier clinical stages.

Analysis of lymphoid and dendritic cells in human lymph node, tonsil and spleen. A study using monoclonal and heterologous antibodies.

The distribution of lymphoid and dendritic cells in human reactive lymph nodes, tonsils and spleens was examined by means of an indirect immunoperoxidase technique, using a panel of monoclonal and heterologous antibodies. The antibodies used were directed against antigens present on T cell subsets (Leu1, leu2a, Leu3a, TA1, OKT6), various types of B cells (BA1, BA2, HLA-DR, CR1) and cells of the mononuclear phagocyte system (alpha HM1, TA1, CR1, OKM1, NA 1/34). In the lymph node and tonsil Leu3a-positive cells (T-helper/inducer phenotype) and Leu2a-positive cells (T-suppressor/cytotoxic phenotype) are found in the thymus-dependent or T-cell area; in the spleen Leu3a-positive cells are found mostly in the periarteriolar lymphocyte sheath (PALS), while Leu2a-positive T-suppressor/cytotoxic cells are almost completely restricted to the cords of Billroth in the red pulp. The cells in the mantle zone of germinal centres and in the primary follicles in lymph nodes, tonsils and spleens have B-cell properties (BA1-, HLA-DR-, and CR1-positive). The cells in the germinal centres show a similar staining pattern (HLA-DR-, and partly CR1-positive). Follicles and T-cell-dependent areas have specific dendritic cells, each with a specific staining pattern: the dendritic reticulum cell (DRC) of the follicle stain with CR1, HLA-DR, BA2 and alpha HM1; the interdigitating cell of the T-cell areas in the lymph node, tonsil and spleen stain with HLA-DR and BA1. Moreover, large dendritic OKT6-positive cells are found in the T-cell areas of some of the peripheral lymph nodes, and are probably Langerhans cells. It is concluded that human lymph nodes and tonsils have an identical compartimentalisation, clearly differing from the spleen in cellular organization.

Intrahepatic distribution of T cell and T cell subsets in cases with type B chronic liver disease by peroxidase-labeled antibody method using monoclonal antibodies.

Intrahepatic distribution of T cell and T cell subsets was studied in 23 cases with type B chronic liver disease (of which 19 cases were also positive for hepatitis B e antigen) and in 6 cases with non-B chronic liver disease by indirect peroxidase-labeled antibody method using monospecific anti-T antibodies and other reagents (anti-Leu series). Hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) in liver tissue were also observed by the peroxidase-labeled antibody method. Membranous expression of HBsAg was found in 16 out of 23 patients with sero-HBsAg, and HBcAg was detected in 15 of them. In these patients, pan-T cells (Leu-1 positive cells) were the predominant cells in the portal tract and the parenchyma. Particularly, T cytotoxic/suppressor cells (Leu-2a positive cells) were often recognized both in sites of piecemeal necrosis and focal necrosis. In some patients whose liver biopsy specimens were obtained during acute exacerbation of chronic hepatitis, T cytotoxic/suppressor cells as well as pan-T cells were increased remarkably in sites of piecemeal necrosis and focal necrosis. These results suggest that T cell cytotoxicity may play an important role in the pathogenesis of liver cell necrosis in type B chronic liver disease.

Immune response to Toxoplasma gondii. I. Toxoplasma-specific proliferation response of peripheral blood lymphocytes from patients with toxoplasmosis.

Peripheral blood leukocytes (PBL) from patients with toxoplasmosis were shown to be highly responsive to in vitro stimulation with Toxoplasma gondii extract as measured by incorporation of [3H]methylated thymidine. Analysis of Toxoplasma-specific proliferative cells in PBL by using monoclonal antibodies specific for human T cell subsets revealed that the Toxoplasma-specific proliferation response of PBL from the patients was mediated by Leu 1, Leu 3a positive cells, that is, helper/inducer T cells. Tests for the Toxoplasma-specific proliferation response may provide a readily available method for the diagnosis of congenital toxoplasmosis, especially during the newborn period.

Phenotypic changes induced in the human thymic all cell line HPB-all by an ingenol ester, milliamin

An ingenol ester, milliamin, induced phenotypic changes in human thymic ALL cell line HPB-ALL. These alterations included morphological changes, increased sRBC-rosetting, the induction of phenotypic changes of differentiation antigens, i.e. a decrease in OKT4-, OKT6-, OKT9-, OKT10- and Leu31-positive cells and an increase in Leu2a-positive cells, growth inhibition and decreased DNA synthesis. These phenotypic changes were qualitatively and quantitatively similar to those induced by other polyol diterpens with tigliane and daphnane hydrocarbon skeletons such as TPA and mezereine, respectively.