Lymphocyte Surface Membrane Research Articles

HMGB1 and Cord Blood: Its Role as Immuno-Adjuvant Factor in Innate Immunity

In newborn the innate immune system provides essential protection during primary infections before the generation of an appropriate adaptive immune response that is initially not fully operative. Innate immune response is evoked and perpetuated by molecules derived from microorganisms or by the damage/death of host cells. These are collectively known as damage-associated molecular-pattern (DAMP) molecules. High-mobility group box 1 protein (HMGB1) or amphoterin, which previously was considered to be only a nuclear factor, has been recently identified as a DAMP molecule. When it is actively secreted by inflammatory cells or passively released from necrotic cells, HMGB1 mediates the response to infection, injury and inflammation, inducing dendritic cells maturation and T helper-1-cell responses. To characterize the role of HMGB1 in the innate and immature defense mechanisms in newborns, human cord blood (CB) mononuclear cells, in comparison to adult peripheral blood (PB) mononuclear cells, have been analyzed for its expression. By flow cytometry and western blot analysis, we observed that in CB and PB cells: i) HMGB1 is expressed on cell surface membranes of myeloid dendritic cell precursors, mostly, and lymphocytes (gamma/delta and CD4+ T cells) to a lesser extent; ii) different pro-inflammatory stimuli or molecules that mimic infection increased cell surface expression of HMGB1 as well as its secretion into extracellular environment; iii) the treatment with synthetic molecules such as aminobisphosphonates (ABs), identified to be γδ T cell antigens, triggered up-regulation of HMGB1 expression on mononuclear cells, as well γδ T lymphocytes, inducing its secretion. The modulation of its secretion and the HMGB1-mediated migration of monocytes indicated HMGB1 as regulator of immune response in an immature system, like CB, through engagement of γδ T lymphocytes and myeloid dendritic cell precursors, essential components of innate immunity. In addition, the increased HMGB1 expression/secretion triggered by ABs, previously characterized for their immuno-modulating and immune-adjuvant capabilities, indicated that immunomodulation might represent a new therapeutical approach for neonatal and adult pathologies.

Amyloidosis associated with chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL), the most common form of leukemia in Western countries, rarely induces glomerular disease, but membranoproliferative glomerulonephritis or immunotactoid glomerulopathy has been reported. The proliferating cells in CLL are of mature B-cell origin and produce monoclonal immunoglobulin (Ig), thus leading to various kinds of autoimmune disorders or immunotactoid glomerulopathy. Although there have been a few reported cases of amyloidosis accompanying CLL, the type of amyloid fibrils has not been demonstrated nor described in detail, particularly regarding monoclonal Ig productivity. We report a rare case of amyloidosis associated with CLL, in which we detected κ-light chain type monoclonal Ig in the sera, urine, and on the surface membrane of lymphocytes, and discuss an association between monoclonal Ig-related disease and non-Hodgkin's lymphoma.

B Cells, Be Gone — B-Cell Depletion in the Treatment of Rheumatoid Arthritis

CD20 protein is expressed on the surface membrane of pre-B lymphocytes and mature B lymphocytes. The protein is located in the B-cell membrane, with 44 amino acids exposed in the extracellular space. Rituximab is a chimeric anti-CD20 antibody whose variable (antigen-binding) region was derived from a mouse antibody so that it has high affinity for CD20. The binding of rituximab to the CD20 antigen interferes with the activation and differentiation of B cells.1 Hematopoietic stem cells, pro-B lymphocytes, and plasma cells do not express CD20. This distribution permits the specific elimination of B cells without preventing the regeneration of B . . .

Blood Lymphocytes, Monocytes and NK Cells Modulate Their Expression of CD44, ICAM‐1, LFA‐1 and MHC Class II After Arrival Into Lymphoid Organs

Adhesion molecules expressed on surface membranes of lymphocytes and other leukocytes enable their entry into the lymphoid and other tissues. However, little is known about molecules that govern the transit of leukocytes through the parenchyma of lymphoid organs proper. We show that in comparison to blood leukocytes, the corresponding cells isolated from lymphoid organs, i.e., lymph nodes and spleen, have a significantly augmented expression of certain surface molecules. The helper and cytotoxic subsets of T cells, as well as B cells, display the increased expression of CD44, ICAM‐1 and LFA‐1, whereas B cells additionally show the augmented expression of MHC class II. In comparison with blood monocytes, splenic monocytes show the increased expression of ICAM‐1 and MHC class II molecules. When compared with blood NK cells, splenic NK cells only show the increased expression of ICAM‐1. The molecules, which we show to be up regulated upon the entry of leukocytes into lymphoid organs, could be involved in their retention within the tissue via cell–cell or cell‐extracellular matrix interactions and in control of their transit through lymphoid tissues.

CD52 antigen as a target for immunotherapy

T he antigenic epitope CD52 is expressed on the surface membranes of peripheral blood lymphocytes, monocytes, and macrophages, as well as the epithelial lining of the male reproductive system. The structural features of the CD52 antigen, specifically its small size, lateral mobility, and closeness to the cell membrane, as well as the sheer abundance of the antigen (CD52 is expressed on 5% of the lymphocyte surface), make it an ideal target for antibody-mediated killing1 (Fig 1). Alemtuzumab (CAMPATH 1H; ILEXTM Oncology, Inc [ILEX], San Antonio, TX), a humanized monoclonal antibody against the CD52 epitope, has a relatively low affinity and requires a concentration of approximately 50 g/mL to achieve maximum binding2 (Fig 2). Nevertheless, it is an extremely potent agent for depletion of lymphocytes in vivo, and a short course of treatment with as little as 40 mg (total) gives rapid and long-lasting depletion of lymphocytes, particularly CD4 cells, in transplant patients.3,4 The potent activity of alemtuzumab appears to be primarily initiated through cell-mediated killing (antibody-dependent cell-mediated cytotoxicity), which is maximal at concentrations as low as 10 ng/mL, whereas optimum activation of complement-mediated lysis requires concentrations of 10 g/mL5 (Fig 3). In patients with chronic lymphocytic leukemia, the indication for which alemtuzumab is approved, both the route of administration and the tumor burden influence the time necessary to achieve peak serum levels. The time to reach peak serum antibody levels is shorter with intravenous administration than with subcutaneous administration, and the blood levels reached after intravenous administration are higher in patients who have a good clinical response. In contrast, serum levels among bone marrow transplant recipients were more consistent among patients, presumably because they did not have a substantial burden of CD52 tumor cells.4 Pharmacokinetic analysis of serum from bone marrow transplant recipients shows that the half-life of alemtuzumab is in the range of 1 to 3 weeks. Therapeutic antibodies have long been used to mediate immunosuppression and prevent acute rejection in transplant recipients. An issue with antibody therapy, however, is the development of antiantibodies that can limit efficacy. Antithymocyte globulin is currently the most commonly used T-cell– depleting agent for induction therapy in kidney transplant recipients. Although antithymocyte globulin has been associated with high graft survival rates ( 98%) and low infection rates ( 10%), the potential for development of xenogeneic antibodies is high ( 78%). Muromonab anti-CD3 (OKT3) is less immunogenic; however, human antimouse antibody development is possible because of its murine origin. Because alemtuzumab is a humanized From the Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom. Address reprint requests to Geoff Hale, PhD, Therapeutic Antibody Centre, Old Road, Headington, Oxford OX3 7JT, United Kingdom. © 2003 Elsevier Inc. All rights reserved. 0955-470X/03/1704-0000$30.00/0 doi:10.1016/S0955-470X(03)00073-9 Figure 1. Structure of the CD52 antigen showing the closeness of the Campath epitope to the lipid anchor. (Modified and reprinted with permission from Hale G. The CD52 antigen and development of the CAMPATH antibodies. Cytotherapy 2001; 3:137. Published by Taylor & Francis.)

γδ T lymphocytes in the peripheral blood of patients with tuberculosis with and without HIV co-infection

Background: Several recent studies suggest that γδ T lymphocytes play an important role in immunity against Mycobacterium tuberculosis. However, the dynamics of these cells in the peripheral blood of patients...

Cell-type specificity of anti-CD45 autoantibodies in systemic lupus erythematosus.

To determine the specificity of anti-CD45 autoantibodies in systemic lupus erythematosus (SLE) for native CD45 and for CD45 expressed by T cells and B cells, and at different stages of cellular activation. CD45 purified from different types of lymphocytes was examined by immunoblotting with sera from patients with SLE. Indirect immunofluorescence experiments were performed with purified anti-CD45 autoantibodies. IgM anti-CD45 autoantibodies in SLE recognize native CD45 expressed on the surface membrane of viable lymphocytes and CD45 purified from activated peripheral T cells and certain T cell lines, but not CD45 purified from B cells or resting peripheral T cells. The presence or absence of reactivity is independent of the individual isoforms expressed. Recognition of CD45 by IgM antilymphocyte autoantibodies in SLE varies with the lineage and state of activation of the lymphocyte target. This pattern of reactivity is consistent with autoantibody specificities involving CD45 glycoforms, rather than CD45 isoforms.

Blood clonal B-cell excess in patients with monoclonal gammopathy of undetermined significance (MGUS): association with malignant transformation.

Fifty-seven patients with monoclonal gammopathy of undetermined significance (MGUS) were analysed for the presence of blood clonal B-cell excess (CBE), defined as a lymphocyte surface membrane kappa/lambda light chain ratio outside the normal range (> 3.5 in kappa-type MGUS and < 0.9 in lambda-type MGUS). 15 patients (26%) had a CBE. The patients were followed for a median time of 8.4 years (range 0.5-20.2). Eight of the 15 CBE+ MGUS patients (53%) developed a B-cell malignancy as compared to 7/42 patients (17%) in the CBE group and the difference in event-free (malignancy-free) observation time was statistically significant (P = 0.01). Cox's regression analysis showed that the presence of CBE was the most powerful predictor of progression to a malignant disease. Two patients with a normal kappa/lambda ratio at first test were analysed repeatedly during follow-up. Subsequently, a CBE appeared which gradually increased in size preceding the clinical diagnosis of a malignant disease.

The Expression of Adhesion Molecules, including CD11a and CD11b, on the Human T Lymphocyte Surface Membrane in Patients with Bronchial Asthma.

The beta-2 integrin consists of lymphocyte function-antigen-1 (LFA-1, CD11a/CD18) which binds to the major ligand intracellular adhesion molecules 1 and 2 (ICAM-1 and ICAM-2), Mac-1 (CD11b/CD18, ligand iC3b) and p150,95 (CD11c/CD18, ligand iC3b). These molecules function not only as adhesion molecules but as signal transduction agents. To investigate the expression of an integrin family in asthma, lymphocyte subsets were assessed using bronchoalveolar lavage fluid (BALF) cells obtained from asthma patients. The relationship between the integrins and lymphocyte subpopulations and their activation state in the BALF was analyzed using two-color flow cytometry in 15 subjects with asthma and in 13 control subjects with chronic bronchitis. The absolute number of CD11a+CD4+ lymphocytes was significantly greater in the asthmatics than in the nonsmoking subjects with chronic bronchitis. In the asthmatics, the expression of the CD11a+CD4+ lymphocytes was significantly higher than the expression of CD11a+CD8+ lymphocytes. The CD2+ lymphocytes had a significant positive regression with the CD11a+CD4+ lymphocytes in the asthmatics. These findings indicate that the CD11a+ antigen is mainly located on the CD4+ lymphocytes in asthmatics, and that the subset of CD11a lymphocytes with the CD4+ antigen increases as the absolute number of T lymphocytes increases. Analysis of BALF revealed the representation of integrins for the ongoing bronchial transfer of information, and may provide an approach to obtain evidence about the inflammatory processes on the surface of the bronchioles in asthma.

Relevant targets for therapy with monoclonal antibodies in allograft transplantation

A 45-year-old woman had been dialyzed for 3 years for end-stage renal disease secondary to polycystic kidney disease. She had received two transfusions and had developed antibodies reacting with 20% of the local screening panel. The patient was offered renal transplantation and agreed to enroll in a randomized study to evaluate the efficacy of a monoclonal antibody (mAb), directed against an adhesion/co-stimulatory molecule, CDIIa, in improving graft survival. In this study, the antibody, which had yielded encouraging results in a recent pilot study, was compared (in an induction protocol) with anti-thymocyte polyclonal globulins. The protocol called for the monoclonal antibody to be administered at 20 mg/day for 10 days, along with 1 mg/kg of steroids (tapered every week, until complete withdrawal at day 45) and 2.5 mg/kg of azathioprine; cyclosporine A (C5A) was begun at the end of the mAb administration. At day 2 post transplantation, the patient excreted 2 liters of urine; her serum creatinine level decreased progressively to levels below 150 j.M (1.6 mg/dl) at day 9. No adverse effect of the mAb was noted. Circulating trough levels reached 7 jsg/ml at day 6 and peaked at 11 jig/mI at day 11. No changes in white blood cell counts were noted after an initial decline of 20% in lymphocyte count. As early as day 3, LFA-1 site occupancy of the patient's lymphocytes by the antibody was saturated. In addition, patient-activated T-cells had lost their capacity to bind a B-cell line bearing ICAM-1, the ligand of LFA-1. An almost total modulation of the patient's CD11a molecule at the lymphocyte surface membrane was found at the end of the treatment. All these biologic effects disappeared by day 30. The patient did not have any rejection episodes during the first 3 months after transplantation; her serum creatinine concentration was 130 jiM (1.4 mg/dl) at that time.

Varying populations of CD59-negative, partly positive, and normally positive blood cells in different cell lineages in peripheral blood of paroxysmal nocturnal hemoglobinuria patients.

CD59-antigen expression on the surface membranes of erythrocytes, granulocytes, monocytes, lymphocytes, and platelets was determined by flow cytometry in 34 healthy controls and 17 patients with paroxysmal nocturnal hemoglobinuria (PNH). In all PNH patients, CD59-negative erythrocytes accounted for > 10% of the total erythrocyte population. Two erythrocyte populations (CD59-negative and normally positive or CD59-negative and partly positive), three populations (CD59-negative, partly positive, and normally positive), and one population (CD59-negative) were demonstrated in ten, six, and one patients, respectively. However, CD59-negative granulocytes did not account for > 10% of the total granulocytes in two patients, and one of them had only a CD59 normally positive granulocyte population. A particular granulocyte population extended over both CD59-negative and partly positive areas was shown in two patients. Two populations (CD59-negative and normally positive) and one population (CD59-negative) were demonstrated in monocytes and lymphocytes. CD59-negative lymphocytes accounted for > 50% of the total lymphocytes in only two patients. Three patients had a CD59 normally positive lymphocyte population. Percentages of CD59-positive platelet population in normal controls were widely various. Therefore, it was usually difficult to discriminate between PNH-affected and normal platelets. Thus, the flow cytometric profiles of CD59-antigen expression varied not only between PNH patients but between cell lineages. The present results and our prior study indicate that CD59 flow cytometry using erythrocytes and granulocytes is most suitable for diagnosing PNH.

CD23 expression in aged rats.

CD23 is considered as an activation marker. Its expression on lymphocytes was studied during aging by using 1- to 142-week-old rats. Before analysis, all animals were injected with monoclonal IgE, in order to detect their full expression of CD23 molecules on their lymphocyte surface membranes. In elderly rats, a decrease was observed in the percentages of CD23+ lymphocytes from mesenteric lymph nodes, spleen, Peyer's patches and peripheral blood. In addition, these lymphocytes expressed significantly fewer CD23 molecules per cell. Almost all CD23+ cells belonged to the B lineage and were bearing both IgM and IgD on their membranes. It is suggested that a decrease in the number of both IgM+IgD+CD23+ lymphocytes and CD23 molecules per cell may contribute to immune deficiency in the elderly.

ブリ末梢血リンパ球上のＣ３レセプターの存在

The presence of C3 receptors on blood lymphocytes of yellowtail Seriola quinqueradiata was investigated by rosette forming assay. The dependence of this adherence reaction on the complement and in particular on the components of the alternative pathway was demonstrated in this experiment. Yellowtail lymphocytes formed rosette with rabbit red blood cells (RRBC) which had been pre-incubated with fresh normal yellowtail serum. This rosette formation was inhibited by treatments of the serum with EDTA, zymosan or heating at 50°C for 20 min. Antibodies with respect to the yellowtail C3 component inhibited the attachment of the sensitised RRBC to the lymphocytes, while antibodies with respect to yellowtail IgM showed no inhibition. On the basis of the above observations, we speculated that the RRBC adherence reaction might depend on C3 component receptors present on the lymphocyte surface membrane. This result indicated the presence of C3 receptors on yellowtail lymphocytes.

Detection of Low Numbers of Lymphocyte Surface Membrane Molecules Using Concentration Immunofluorescence Analysis (CIA)

Using viable lymphocytes in a concentration immunofluorescence assay (CIA), conditions were ascertained which allow detection of low numbers of surface membrane molecules. Utilizing 3 layer labeling [monoclonal antibody (MAb) specific for the membrane molecule, biotin-conjugated antibody specific for the MAb, and R-phycoerythrin-strepavidin], large numbers of lymphocytes in the assay wells, and the Pandex Fluorescence Concentration Analyzer, a fluorescence signal significantly above background was generated by as few as 8 x 10(7) molecules among 4 x 10(5) lymphocytes. Experiments using membrane molecules (Fc gamma R II, Ia antigens, Ly-39) which differ considerably in their level of expression indicated that comparable signals were generated by equivalent numbers of labeled molecules in a cell population irrespective of the number of molecules on an individual cell. Thus, CIA is theoretically capable of detecting membrane molecules whose expression is as low as 2 x 10(2) per cell. CIA should be useful in the assay of cytokine receptors and other lymphocyte membrane molecules expressed at low levels, and in the development of MAb specific for these molecules.

Interactions between murine B lymphocyte surface membrane molecules. Loaded but not free receptors for complement and the Fc portion of IgG co-cap independently with cross-linked surface Ig.

We have investigated the possible physical interactions between CR, receptors for the Fc gamma R and surface Ig (sIg) on the surface membrane of murine B lymphocytes. We used the rat mAb to murine CR, 8C12, and 7G6, as CR ligands, and soluble Ag-antibody complexes as FcR ligands; and F(ab')2 fragments of rabbit antibodies specific for mouse IgM and IgD as sIg ligands. We have found that: 1) sIg, CR, and Fc gamma R are not directly linked, because capping of any one did not affect the expression of the others; 2) the mAb 8C12 and 7G6 failed by themselves to cross-link CR; 3) soluble Ag-antibody complexes crosslinked some, Fc gamma R on a minority of Fc gamma R+ lymphocytes; 4) once loaded with anti-CR mAb, CR co-capped with sIg when sIg was cross-linked; 5) once loaded with Ag-antibody complexes, Fc gamma R also co-capped with sIg when sIg was sIg was cross-linked; 6) loading of Fc gamma R did not affect the co-capping of surface CR with cross-linked sIg and conversely, loading of CR did not affect the co-capping of Fc gamma R with cross-linked sIg; only loaded CR or Fc gamma R co-capped with sIg regardless of the status of the other surface molecule; 7) neither loaded nor free CR co-capped with cross-linked Fc gamma R, and neither loaded nor free Fc gamma R co-capped with cross-linked CR. These results demonstrate that both Fc gamma R and CR independently become associated with sIg when either receptor is loaded and sIg is cross-linked.

Recombinant bovine interferon-gamma enhances expression of class I and class II bovine lymphocyte antigens

Recombinant bovine interferon-gamma augments expression of class I and class II histocompatibility antigens on the surface membrane of bovine lymphocytes. Immunofluorescence techniques using a series of monoclonal anti-HLA antibodies demonstrate that this enhancement is detectable as early as 24 h after incubation with rBoIFN, while maximum surface expression is obtained within 3–5 days. A concentration as low as 10 units of rBoIFN is effective. Such results may be useful for characterizing the BoLA gene products.

Immunoelectron microscopic demonstration of antigenic sites on lymphoid cells using a human monoclonal antibody (Ha6D3).

The reactivity of a human monoclonal antibody directed against human B and T lymphocytes was tested for the first time at the ultrastructural level. The antigenic sites detected by this antibody were localized on the surface membrane of lymphocytes and, to a lesser extent, in the cytoplasm on membranes of the endoplasmic reticulum and perinuclear envelop of some centroblasts and immunoblasts. Ultrastructural demonstration of target antigen detected by human monoclonal antibodies may be important prior to therapeutic application of these antibodies.

Monoclonal anti-IL2-receptor in organ transplantation.

So far, only monoclonal antibodies directed at functional target molecules on lymphocyte surface membrane have been proven useful in preventing or reversing allograft rejection episodes. Antibodies directed against light chain (P55) of interleukin 2 (IL2) receptor and able to interfere with IL2 binding on the IL2-receptor in its high-affinity conformation (only expressed on activated T cells) are effective in various animal models and recently in a preventive protocol in human kidney graft recipients. Thus, IL2-receptor targeting emerges as a new therapeutic strategy involving only a small pool of progenitors committed against donor antigens. Furthermore, membranous or soluble forms of P55, as indicators of the presence of alloreactive clones in the graft or at the peripheral level, may offer new tools for monitoring the rejection process.

Monoclonal anti-IL2-receptor in organ transplantation

Abstract. So far, only monoclonal antibodies directed at functional target molecules on lymphocyte surface membrane have been proven useful in preventing or reversing allograft rejection episodes. Antibodies directed against light chain (P55) of interleukin 2 (IL2) receptor and able to interfere with IL2 binding on the IL2-receptor in its high-affinity conformation (only expressed on activated T cells) are effective in various animal models and recently in a preventive protocol in human kidney graft recipients. Thus, IL2-receptor targeting emerges as a new therapeutic strategy involving only a small pool of progenitors committed against donor antigens. Furthermore, membranous or soluble forms of P55, as indicators of the presence of alloreactive clones in the graft or at the peripheral level, may offer new tools for monitoring the rejection process.

Inhibition of murine lymphocyte proliferation by the B subunit of cholera toxin.

Cholera toxin is known to inhibit lymphocyte activation in vitro, an effect attributed to its ability to activate adenylate cyclase and increase intracellular cyclic adenosine monophosphate. In these studies the effects of both cholera toxin (CT) and its purified binding subunit (CT-B) on lymphocyte proliferation in vitro was examined, using a variety of cell activators. We found that both CT and CT-B inhibited mitogen- and antigen-induced T cell proliferation and anti-IgM-induced B cell proliferation in a dose-dependent manner. However, only CT-inhibited lipopolysaccharide-induced B cell proliferation. Neither CT nor CT-B inhibited antigen uptake and presentation by macrophages. The CT-B preparation used was shown not to activate lymphocyte adenylate cyclase, although CT itself was a strong activator of this enzyme. Both molecules had to bind to the lymphocyte surface in order to inhibit. The time course of inhibition of both CT and CT-B was similar in that either could be added up to 24 hr after culture initiation and still inhibit substantially. The addition of excess human recombinant interleukin 2 to the cultures did not affect the inhibition by CT, and had only a partial affect on inhibition by CT-B. Similarly, CT was able to substantially inhibit recombinant interleukin 2-dependent T lymphoblast proliferation, whereas CT-B had only a small inhibitory effect. Inhibition was not major histocompatibility complex-restricted. We conclude that the binding of CT or CT-B to the lymphocyte surface membrane interferes in some way with the activation mechanism leading to proliferation. The inhibition mediated by CT-B does not involve the stimulation of intracellular adenylate cyclase. CT appears to inhibit both by binding via its B subunit and by activation of adenylate cyclase via its A subunit.