IL-10 production by CD19(+)CD5(+) B cells was investigated, by determining the expression levels of CD19, a classical B cell marker. Peripheral mononuclear cells were stained with fluorescence-conjugated anti-CD5, anti-CD19, anti-IL-10, and Annexin V. Interestingly, IL-10-producing B cells were found to be localised within the CD19(low)CD5(+) B cell subset. Apoptotic changes were also observed mainly in CD19(low) cells among B cells. Thus, CD5(+) B cells should be classified as CD19(high) and CD19(low) cells, and the immunological significance of CD19 for the IL-10 production by CD5(+) B cells requires further studies.
Antigens, CD19/metabolism ; Antigens, CD5/metabolism ; Apoptosis/immunology ; B-Lymphocyte Subsets/cytology/*immunology ; Cell Separation ; Flow Cytometry ; Humans ; Interleukin-10/*biosynthesis

Myasthenia gravis is one of the typical organ specific autoimmune disease and the CD5+ B-lymphocytes are known to be associated with the secretion of autoimmune antibodies. The authors performed the study to establish an animal model of experimental autoimmune myasthenia gravis (EAMG) by immunizing the nicotinic acetylcholine receptor (AChR) and to understand CD5+ B-lymphocyte changes in peripheral blood of EAMGs. Lewis rats weighing 150-200 g were injected subcutaneously three times with 50 microg AChR purified from the electric organ of Torpedo marmorata and Freund's adjuvant. The EAMG induction was assessed by evaluating clinical manifestations. The CD5+ B-lymphocyte was double stained using monoclonal PE conjugated anti-CD5+ and FITC conjugated anti-rat CD45R antibodies and calculated using a fluorescence-activated cell sorter (FACS). In three out of ten Lewis rats injected with purified AChR, the EAMG models were established. The animals showed definite clinical weakness responded to neostigmine; they had difficulty in climbing the slope, or easily fell down from a vertical cage. The range of CD5+ B-lymphocytes of peripheral blood in the EAMG models was 10.2%-17.5%, which was higher than in controls. In conclusion, the EAMG models were successfully established and the CD5+ B-lymphocyte expression in peripheral blood increased in EAMGs. This provided indirect evidence of the autoimmune pathomechanism of human myasthenia gravis.
Animal ; Antigens, CD5/immunology* ; B-Lymphocytes/immunology* ; Disease Models, Animal ; Myasthenia Gravis/immunology* ; Myasthenia Gravis/chemically induced ; Rats ; Rats, Inbred Lew

We investigated the changes of CD5+ B cells in the peripheral blood of 20 Kawasaki disease (KD) patients. The percentage of CD5+ B cells in the total lymphocytes and in the total B cells significantly decreased during the acute phase of KD(p< 0.01), compared to that in the age-matched normal control subjects. After intravenous immunoglobulin(IVIG) treatment, the percentage of CD5+ B cells increased, but was still lower than that in the normal controls(p< 0.01). During the convalescent phase of the disease, the percentage of CD5+ B cells was restored to the normal levels. The levels of CD5+ B cell percentage in the total B cells of the patients with acute febrile disease showed similar levels to age-matched normal controls. The decreased CD5+ B cells in the patients with KD provides an additional abnormal immunological finding during the acute phase of the disease.
Acute Disease ; Antigens, CD5/*analysis ; B-Lymphocytes/*immunology ; Child ; Child Preschool ; Female ; Human ; Infant ; Male ; Mucocutaneous Lymph Node Syndrome/*immunology/physiopathology ; Reference Values

OBJECTIVETo investigate the number of peripheral blood CD5(+) B cells and their ability of secreting IL-10 in patients with immune thrombocytopenia (ITP).

METHODSPeripheral blood lymphocytes were isolated from 57 pre-treated, 40 post-treated ITP patients and 25 controls using Ficoll-Hypaque density centrifugation and then stained with PE-CD5/FITC-CD19 for flow cytometric analysis. After 24-hour culture, lymphocytes were stained with APC-IL-10 for intracellular cytokine detection. ELISA assay was employed to determine IL-10 concentration in supernatants.

RESULTSThe percentage and absolute number of CD5(+) B cells in peripheral blood from pre-treated ITP patients were significantly higher than that from normal controls (3.75 ± 2.37)% vs (2.10 ± 1.08)%, P < 0.01; (6.29 ± 5.77)× 10(7)/L vs (3.06 ± 1.90)× 10(7)/L, P < 0.01. CD5(+) B cells expressed more intracellular IL-10 than other lymphocyte subsets both in ITP patients and normal controls. The percentages of IL-10(+) cells within CD5(+) B cells in pre-treated ITP patients and normal controls were (29.51 ± 20.73)% and(15.90 ± 9.58)%, respectively(P < 0.01). Intracellular mean fluorescence intensity (MFI) of IL-10 in CD5(+) B cells was 27.95 ± 13.99 in pre-treated patients, which was significantly higher than that in controls (P < 0.01). In contrast, IL-10 concentration in supernatants was (173.05 ± 102.50) ng/L in pre-treated ITP group, which was lower than that (230.61 ± 76.96) ng/L in controls. In patients who achieved remission, the number of CD5(+) B cells decreased to level comparable to normal controls. While intracellular IL-10 MFI of CD5(+) B cells in post-treated ITP patients remained as high as in pre-treated ones, the IL-10 concentration in supernatants increased to level similar to controls.

CONCLUSIONThe significantly increased number of CD5(+) B cells and accumulated IL-10 in CD5(+) B cells suggested impaired IL-10 secretion in ITP patients. The number and the ability of secreting IL-10 of CD5(+) B cells could be restored after effective treatments in patients with ITP.

Adult ; Aged ; B-Lymphocytes ; immunology ; metabolism ; CD5 Antigens ; metabolism ; Case-Control Studies ; Female ; Humans ; Interleukin-10 ; blood ; Male ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; blood ; immunology ; Young Adult

OBJECTIVETo study the possible loss of pan-T cell antigens CD2, CD3, CD5 and CD7 in Kikuchi's disease and to evaluate the role of T cell antigen loss in distinguishing benign from malignant T-cell lymphoid lesions.

METHODSFormalin-fixed and paraffin-embedded tissues of 33 cases of Kikuchi's disease and 15 cases of reactive lymphoid hyperplasia were studied by EliVision immunohistochemical staining for CD2, CD3, CD5 and CD7.

RESULTSTwenty-four of the 33 (72.7%) cases of Kikuchi's disease lost one or more of the pan-T cell antigens, including the loss of CD5 only (13 cases), CD7 only (1 case), CD2 only (1 case), CD2 and CD7 (2 cases), CD5 and CD7 (4 cases), CD2 and CD5 (2 cases), and CD2, CD7 and CD5 (1 case). Amongst these cases, the commonest antigen loss was CD5 (20 cases, 60.6%), followed by CD7 (8 cases, 24.2%) and CD2 (6 cases, 18.2%). Compared with the xanthomatous subtype of Kikuchi's disease, the loss of antigens was more commonly seen in the proliferative and necrotizing subtypes. Analysis of follow-up data showed that the loss of antigens in Kikuchi's disease was not significantly associated with the prognosis. In reactive lymphoid hyperplasia, the expression of CD2, CD3, CD5 and CD7 was seen in all cases with similar intensity, with no obvious pan-T cell antigen loss.

CONCLUSIONLoss of one or more pan-T cell antigens in Kikuchi's disease is demonstrated in present study, suggesting that the immunophenotypic pattern is not unique in T cell lymphoma.

Adolescent ; Adult ; Antigens, CD7 ; metabolism ; CD2 Antigens ; metabolism ; CD3 Complex ; metabolism ; CD5 Antigens ; metabolism ; Child ; Child, Preschool ; Female ; Follow-Up Studies ; Histiocytic Necrotizing Lymphadenitis ; immunology ; pathology ; Humans ; Male ; Middle Aged ; Pseudolymphoma ; immunology ; Recurrence ; T-Lymphocytes ; immunology ; Young Adult

The aim of study was to investigate the lymphocyte changes in peripheral blood of patients with Graves's disease accompanied by hematocytopenia and to explore its pathogenesis. The quantity and ratio of Th(2), Th(1), CD5(+) B, Bcl-2 level in 24 Graves's disease patients with hematocytopenia were detected by FACS, and 18 adults were selected as normal controls. The results indicated that the percentages of Th(1), Th(2), ratio of Th(2)/Th(1), CD5(+) B, Bcl-2 level in peripheral blood of the patients were (0.81 +/- 0.45)%, (6.83 +/- 3.02)%, (20.55 +/- 6.15)%, (20.89 +/- 1.62)%, (80.25 +/- 15.56)%, respectively, and were higher than those of normal controls [(0.39 +/- 0.24)% (P<0.05), (0.28 +/- 0.15)% (P<0.01), (0.52 +/- 0.12)% (P<0.01), (7.89 +/- 0.38)% (P<0.05), (36.49 +/- 6.79)% (P<0.05)]. It is concluded that the pathogenesis of Graves's disease with hematocytopenia may be related to unbalance of Th(1)/Th(2), increase of Th(2) inducing over-expression of CD5(+) B and Bcl-2 on B cell.
Adolescent ; Adult ; B-Lymphocytes ; immunology ; metabolism ; CD5 Antigens ; immunology ; Female ; Graves Disease ; complications ; immunology ; Humans ; Male ; Middle Aged ; Pancytopenia ; complications ; immunology ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; Th1 Cells ; immunology ; Th2 Cells ; immunology

OBJECTIVETo investigate the percentage of splenic CD(5)(+) B lymphocytes in chronic idiopathic thrombocytopenic purpura (IT) and the impact of splenic CD(5)(+) and CD(5)(-) B lymphocytes on the production of platelet glycoprotein (GP)-specific autoantibodies.

METHODSSplenic CD(5)(+) B lymphocytes were identified by two-color flow cytometric analysis in eight patients. Four of the eight patients displayed plasma autoantibodies against both GPIIb/IIIa and GPIb/IX, and their splenic B lymphocytes were separated by Ficoll-Hypaque density gradient and sheep erythrocyte, and further purified by magnetic activate cell separation (MACS). Purified CD(5)(+) and CD(5)(-) B lymphocytes were cultured separately with or without staphylococcus aureus cowan I (SAC). GP specific autoantibodies in culture supernatants were measured by modified monoclonal antibody immobilization of platelet antigen assay (MAIPA).

RESULTSThe percentage of splenic CD(5)(+) B lymphocytes in ITP patients was slightly higher than that in control with no statistical significance. MACS purified splenic CD(5)(+) and CD(5)(-) B lymphocytes from three out of four ITP patients produced high levels of anti-GPIIb/IIIa and anti-GPIb/IX antibodies. Culture supernatants of CD(5)(+) B lymphocytes from the other patient showed positive reaction only in GPIb/IX MAIPA. Culture supernatant of CD(5)(-)B lymphocytes from the same patient were double positive in both GPIIb/IIIa and GPIb/IX MAIPA.

CONCLUSIONSBoth splenic CD(5)(+) and CD(5)(-) B lymphocytes produce platelet GP-specific autoantibodies in chronic ITP with similar antibody spectrum and titer, and may all play a role in the autoimmune pathogenesis of ITP.

Adolescent ; Adult ; Autoantibodies ; biosynthesis ; B-Lymphocytes ; immunology ; CD5 Antigens ; analysis ; Chronic Disease ; Female ; Humans ; Male ; Middle Aged ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Platelet Glycoprotein GPIb-IX Complex ; immunology ; Purpura, Thrombocytopenic, Idiopathic ; immunology ; Spleen ; cytology

Calreticulin (CRT) is a multifunctional molecule in both intracellular and extracellular environment. We have previously found that a recombinant CRT fragment (rCRT/39-272) could modulate T cell-mediated immunity in mice via activation and expansion of CD1d(hi)CD5⁺ B cells as well as induction of CRT-specific regulatory antibodies. Antibody secreting cells (ASCs) are terminally differentiated B cells responsible for producing antibodies to participate in positive immune response as well as immune regulation. In this study, we demonstrate that rCRT/39-272 differentiates murine CD1d(hi)CD5⁺ B cells into ASCs marked by increased expression of plasma cell-associated transcription factors and production of polyreactive antibodies against DNA and CRT in vitro. Intraperitoneal administration of rCRT/39-272 augmented differentiation of CD1d(hi)CD5⁺ B cells into ASCs in naïve mice or mice with experimental autoimmune encephalomyelitis. Thus, we propose that ASC differentiation and subsequent antibody production of CD1d(hi)CD5⁺ B cells are key steps in CRT-mediated immunoregulation on inflammatory T cell responses.
Animals ; Antigens, CD1d ; metabolism ; Autoantibodies ; biosynthesis ; B-Lymphocytes ; cytology ; drug effects ; immunology ; metabolism ; CD5 Antigens ; metabolism ; Calreticulin ; chemistry ; Cell Differentiation ; drug effects ; Encephalomyelitis, Autoimmune, Experimental ; immunology ; Humans ; Mice ; Peptide Fragments ; chemistry ; pharmacology ; Solubility

To establish reference values of various immunophenotypic markers in B lymphocyte population in healthy Chinese adults and build background information for accurate interpretation of B cell immunophenotyping data in clinical practice, peripheral blood from 41 healthy adults were collected separately into test tubes containing EDTA-K(2) and stored in room temperature no more than 24 hours before analysis. Whole blood lysis technique and multiparameter flow cytometry were applied to immunophenotype B cells gated on CD19/SSC dot-plot. The results showed that CD22, CD20, CD62L, CD40, CD24, CD79b, CD79a, and FMC-7 were almost positive in the circulating B cell population, whereas CD11a, CD80, CD103, CD10, CD40L, CD54, CD95L, CD86, and CD95 were almost negative in the peripheral blood B lymphocytes. CD18, CD44, CD23, CD5, CD11c and CD43 were positive in different B cell subpopulations. 78% of B cells were IgD positive and ratio kappa/lambda was 1.26. The significance of all these markers in the differential diagnosis of lymphoproliferative diseases was discussed. The conclusion is that it is necessary to consider the qualitative and quantitative levels of expression of various markers in normal B cell population in order to accurately interpret the pathological immunophenotypic data in clinical practice. It is also important to note the immunotypic differences of B cells between Chinese and Western populations.
Adult ; Aged ; B-Lymphocytes ; immunology ; CD5 Antigens ; analysis ; Female ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Intercellular Adhesion Molecule-1 ; analysis ; Leukemia, Lymphocytic, Chronic, B-Cell ; immunology ; Lymphoma ; immunology ; Male ; Middle Aged ; Receptors, IgE ; analysis

Fine-needle aspiration (FNA) of lymph nodes has been regarded as a useful method in the diagnosis of lymphadenopathy. However, this procedure has been shown to be of limited value in the diagnosis of low or intermediate grade malignant lymphomas in some studies. Immunophenotyping is an essential adjunct to cytomorphology for the diagnosis of lymphoma by FNA. Immunophenotyping using flow cytometry (FCM) is rapid, objective and reliable. Using FCM, multiparametric analysis of 33 FNA materials from lymph nodes was performed and profiles of surface markers of lymphoid cells were assessed. In reactive hyperplasia, patterns of cell surface markers were quite variable, but disclosed polyclonality. Most of the B-cell lymphomas showed immunophenotypes for B-cell lineages with their kappa: lambda or lambda: kappa ratio being over 3:1. In T-cell lymphomas, T-cell surface markers were predominantly expressed as well. In conclusion, our results suggest that immunophenotyping of lymph node aspirates is a valuable diagnostic adjunct for lymphoproliferative disorders, particularly in B-cell lymphomas because immunophenotyping can be easily and adequately performed by FCM.
Antigens, CD19/analysis ; Antigens, CD20/analysis ; Antigens, CD3/analysis ; Antigens, CD4/analysis ; Antigens, CD5/analysis ; Antigens, CD7/analysis ; Antigens, CD8/analysis ; B-Lymphocytes/immunology ; B-Lymphocytes/chemistry ; Biopsy, Needle ; Flow Cytometry/methods* ; Hodgkin Disease/pathology ; Human ; Immunophenotyping ; Lymph Nodes/pathology ; Lymph Nodes/chemistry ; Lymphatic Diseases/pathology* ; Lymphatic Metastasis/pathology ; Lymphoma, B-Cell/pathology* ; Lymphoma, Non-Hodgkin/pathology ; T-Lymphocytes/immunology ; T-Lymphocytes/chemistryt