Subset specificity of antilymphocyte antibodies in systemic lupus erythematosus

Using indirect immunofluorescence and flow cytometry, we determined the proportion and number of T3+, T4+, and T8+ cells in the peripheral blood of patients with systemic lupus erythematosus whose sera were positive for cold-reactive antilymphocyte antibodies versus values in patients whose sera were negative for these antibodies. There was a disproportionate reduction in T4+ peripheral lymphocytes when cold-reactive antilymphocyte antibodies preferentially cytotoxic for this subpopulation were present in autologous serum. The decrease in this subset was responsible for a reduction in the T4:T8 ratio; variation in the number and proportion of T8+ cells was insignificant. A similar, but autoantibody-independent, alteration in the T4+ subpopulation was found in patients who were receiving prednisone therapy. A relationship between T cell population abnormalities and systemic lupus erythematosus disease activity, per se, was not observed.     

Subset specificity of antilymhocyte antibodies in systemic lupus erythematosus. II. Preferential reactivity with T4 + cells is associated with relative depletion of autologous T4 + cells

Using indirect immunofluorescence and flow cytometry, we determined the proportion and number of T3+, T4+, and T8+ cells in the peripheral blood of patients with systemic lupus erythematosus whose sera were positive for cold-reactive antilymphocyte antibodies versus values in patients whose sera were negative for these antibodies. There was a disproportionate reduction in T4+ peripheral lymphocytes when cold-reactive antilymphocyte antibodies preferentially cytotoxic for this subpopulation were present in autologous serum. The decrease in this subset was responsible for a reduction in the T4:T8 ratio; variation in the number and proportion of T8+ cells was insignificant. A similar, but autoantibody-independent, alteration in the T4+ subpopulation was found in patients who were receiving prednisone therapy. A relationship between T cell population abnormalities and systemic lupus erythematosus disease activity, per se, was not observed.     

Cold reactive lymphocytotoxic activity in autoimmune thyroid disease

An increased incidence of cold-reactive lymphocytotoxic activity (LCTA) has been demonstrated in the sera of patients with autoimmune thyroid disease. Twenty-six of 79 (33%) patients with Graves' disease and 9 of 21 (43%) patients with Hashimoto's thyroiditis had cold-reactive LCTA detected by microcytotoxicity assay compared to 6 of 42 (14%) normal controls. There was no correlation between LCTA and age, sex, MCHA titre or TGHA titre. A positive correlation with FTI and LCTA in Hashimoto's patients was demonstrated, but no such correlation was demonstrable in Graves' patients. The lymphocytotoxic activity was directed preferentially against B cells. There was no preferential lysis of T-cell subsets as defined by monoclonal antibodies, and the lymphocytotoxins were equally reactive with normal lymphocytes and toxic Graves' lymphocytes. The significance of cold-reactive lymphocytotoxic activity in the pathogenesis of autoimmune thyroid disease remains to be determined.     

A preliminary note on the composition of lymphocytes in human peripheral lymph.

Pilot experiments on the composition of lymphocytes in human peripheral lymph, mainly from healthy human volunteers, are presented. The experiments undertaken so far show a reduced proportion of B cells and an increased proportion of "Ia" positive lymphocytes in peripheral lymph as compared to peripheral blood. If confirmed, these data would suggest that lymphocyte circulation through extravascular subcutaneous tissues is a highly selective process.     

CD8+ T lymphocytes of African green monkeys secrete an immunodeficiency virus-suppressing lymphokine.

Following natural and experimental infection by simian immunodeficiency virus SIVagm of African green monkeys (AGMs), the natural host, there is no evidence for the development of an immunodeficiency. Within the framework of our studies on human immunodeficiency virus (HIV)/SIV pathogenesis, we investigated the influence of CD8 T lymphocytes on SIVagm replication in AGM CD4 T lymphocytes in vitro. The following observations were made: (i) Peripheral blood mononuclear cells from both seronegative and seropositive AGMs contained only a low proportion (i.e., 10%) of CD4+ lymphocytes, whereas a high proportion (80%) of CD8+ cells was detected. Even after persistent SIVagm infection, CD4 T lymphocytes do not decrease in number. (ii) The target of in vitro infection of peripheral blood cells is the CD4+ mononuclear cell (T lymphocytes, monocytes) and SIVagm infects by binding to the CD4 molecule. (iii) In both naturally and experimentally SIVagm-infected AGMs the CD4+ T cells and monocytes, but not the CD8+ T cells, harbor DNA provirus. (iv) Virus reisolation and virus replication of SIVagm in CD4 T lymphocytes from seropositive AGMs is suppressed in the presence of autologous CD8 T lymphocytes or a soluble factor produced by these cells. Taken together, one possible reason for the apathogenicity of the SIVagm infection in AGMs may be the suppression of virus replication by a soluble, yet unidentified factor secreted by CD8 lymphocytes quantitatively dominating among peripheral blood cell populations. We have tentatively termed this factor "immunodeficiency virus-suppressing lymphokine." In addition, we show that immunodeficiency virus-suppressing lymphokine from AGMs is able to suppress HIV-1 replication in human CD4+ T cells.     

Specificity analysis of human monoclonal antibodies reactive with cell surface and intracellular antigens.

Over 4350 human Ig-secreting hybrids have been generated through the fusion of human lymphocytes with NS-1 (mouse), LICR-2, SKO-007, GM4672, or UC729-6 (human) myeloma and lymphoblastoid cell lines. NS-1 proved to be the most satisfactory fusion partner, and 83% of the stable Ig-secreting clones were derived from NS-1 fusions. Three hundred five hybrids produced human monoclonal antibodies (hmAb) reactive with cell surface or intracellular antigens expressed by cultured human tumor cell lines, and 111 of these have undergone detailed serological specificity analysis. Several general points have emerged from our study of hmAb: A significant proportion of the human B-cell clones produce antibody reactive with cellular antigens. The majority of these antigens have an intracellular location and are broadly distributed. Intracellular and cell surface differentiation antigens and other antigens with restricted distribution have been defined by hmAb, including two cell surface antigens not detected on normal cells. The relationship of these findings to cancer is unclear, as hmAb reactive with antigens showing distinctive distribution have been generated from the lymphocytes of normal individuals as well as tumor-bearing patients.     

Association of cold-reactive antilymphocyte antibodies with lymphopenia in systemic lupus erythematosus

In a prospective study 26 of 29 patients with systemic lupus erythematosus had cold-reactive antilymphocyte antibodies cytotoxic for autologous lymphocytes and lymphocytes from normal subjects. The level of antilymphocyte antibodies was highly correlated, by linear regression analysis, with lymphopenia in these patients. The data suggested that both the avidity and the concentration of these antibodies were important determinants in this relationship. A clear association between increased antilymphocyte antibody activity and exacerbation of SLE was demonstrated. Apart from lymphopenia, however, neither type of clinical manifestation nor any particular serologic abnormality appeared to be related to the presence of antilymphocyte antibodies.     

The potential immunosuppressive effect of bilirubin

The effect of bilirubin on the cells of the immune system was studied using various in vitro and in vivo systems. Bilirubin was found to inhibit the in vitro chemotactic (migratory) activity of human granulocytes; on the other hand, a temporary stimulation of phagocytic activity of both granulocytes and monocytes was found in mice injected with a single i.p. injection of bilirubin. Continuous i.v. infusion of bilirubin produced a significant decrease of antibody (plaque) forming cells in the spleens of mice immunized with sheep red blood cells. In newborns suffering from hyperbilirubinaemia, enhanced proportion of "activated" lymphocytes occurred. Finally, bilirubin possessed a significant in vitro cytotoxic effect in human adult and newborn blood lymphocytes and granulocytes. A brief survey of so far known immunological effects of bilirubin is discussed.     

Differences in T cell subsets between men and women with idiopathic thrombocytopenic purpura

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder, occurring predominantly in women. We studied by flow cytofluorimetry the T cell subsets in men and women with ITP and compared them with healthy sex-matched volunteers. In healthy controls, women were found to have higher proportions of T helper/inducer (Th/i) and lower T suppressor/cytotoxic (Ts/c) lymphocytes and consequently higher Th/i:Ts/c ratios than men. Accordingly, in clinical surveys, patients and controls should be matched for sex for proper comparisons. In patients with ITP in its active phase, an imbalance in T cell subsets was found in both sexes. The perturbation was more severe in women who had a marked decrease in number and proportion of Th/i lymphocytes and an increase in the proportion of Ts/c lymphocytes, whereas in men only, the proportion of Th/i lymphocytes was decreased. When patients with active disease were compared to those with ITP in remission, the decrease in Th/i subsets still persisted in both sexes but the Ts/c subset in women had returned to normal proportions. Therefore, the immune imbalance in ITP is more marked in women than men; imbalances in both Th/i and Ts/c are present in women while Ts/c appears not to be involved in men.     

Inhibition of neutrophil responses by cyclosporin A. An insight into molecular mechanisms

OBJECTIVE: Cyclosporin A (CsA) is an effective agent in rheumatoid arthritis (RA), slowing joint damage progression. Its therapeutic effect on T lymphocytes has been studied extensively, but there is little information available about neutrophils, the cells responsible for a substantial proportion of inflammation. A study was performed to investigate the in vitro effects of CsA on neutrophil functions triggered by several agonists and determine whether the drug could counteract the binding of formyl-methionyl-leucyl-phenylalanine (fMLP) to its receptor and/or modulate changes in the intracellular Ca(2+) concentration ([Ca(2+)]i). METHODS: CsA was added to neutrophils 5-50 min before the incubation steps for neutrophil function assays (chemotaxis, superoxide anion production, lysozyme release), calcium measurements and receptor binding experiments. RESULTS: CsA appeared to be particularly effective in lowering chemotaxis, superoxide anion production and lysozyme release induced by different agonists. However, it did not significantly affect either basal or agonist-stimulated neutrophil [Ca(2+)]i and the interaction between fMLP and its receptor. CONCLUSIONS: Because of its in vitro inhibition of neutrophil functions, CsA appears to have considerable potential as an anti-inflammatory drug. Moreover, as it is also a potent immunosuppressive agent, it may reduce the progression of joint damage in RA. More work remains to be done to clarify the molecular mechanism of CsA action on neutrophils.     

Autologous and allogeneic suppressor lymphocyte inhibition of human erythroid colony forming unit proliferation

Certain in vitro and in vivo animal studies have supported the concept that lymphocyte-derived microenvironmental factors are important in erythroid proliferation. Considerable controversy has developed regarding the applicability of these concepts to human erythroid proliferation. We, therefore, evaluated the role of lymphocytes on human erythroid colony forming unit (CFUE) proliferation in the plasma clot system. Bone marrow (BM) was obtained from normal donors and cocultured with the following cell populations: a) cultured (6 day) lymphocytes autologous or allogeneic to BM; b) cultured lymphocytes stimulated with conconavalin A (Con A), allogeneic lymphocytes or streptokinase streptodornase (SKSD). In general, unstimulated cultured lymphocytes enhanced CFUE proliferation. In contrast, lymphocytes stimulated with Con A. SKSD, or allogeneic lymphocytes suppressed lymphocyte proliferation. The suppressor cell was concentrated in the T-cell fraction obtained by sheep red blood cell rosetting followed by ficoll-Hypaque centrifugation. Both allogeneic and autologous stimulated T-cells suppressed CFUE. Moreover, supernatants from stimulated lymphocyte cultures suppressed CFUE proliferation although the cell of origin and characteristics of the suppressive factors have not been defined. These data support the concept that lymphocytes may play an important role in modulating the human BM erythroid microenvironment.     

Cold hemagglutinin disease associated with IgG cold-reactive antibody

Six patients with chronic idiopathic cold hemagglutinin disease were studied whose serum cold agglutinin was not inactivated or was incompletely inactivated with the IgM-reducing agent dithiothreitol. In five of these patients, isolation of the antibodies revealed that two patients had predominantly IgG cold-reactive antibody, which was associated with smaller amounts of IgM in one patient and with IgA in the other; two patients had predominantly IgM cold agglutinin with lesser amounts of cold-reactive IgG; and one patient had an IgG cold agglutinin only. Both patients with predominantly IgG cold-reactive antibodies were treated with splenectomy and subsequently had a rise in more than hemoglobin levels that has been maintained for over 36 months without additional therapy. Two of the other three patients were treated with glucocorticoids only and responded similarly. These data indicate that a subset of patients with cold hemagglutinin disease have IgG cold-reactive antibodies. In contrast to patients with typical cold agglutinin disease, this subset appears responsive to glucocorticoids and splenectomy.     

A monoclonal antibody reactive with a subset of human plasma cells

A mouse monoclonal antibody (R1-3) raised against a human plasmacytoma xenograft and reactive with human plasma cells is described. The antibody reacts with a subset of plasma cells and lymphocytes, but is nonreactive with granulocytes, normoblasts, thymus, spleen or T lymphocytes. Flow cytometer double labelling experiments showed that approximately 50% of R1-3 positive bone marrow cells expressed surface immunoglobulin, but no R1-3 positive cells also expressed B1 or Leu 4. In a myeloma patient the R1-3 reactive cells were found to have a higher rate of DNA proliferation and were aneuploid as determined by flow cytometry. The R1-3 antigen is expressed on late B lymphocytes and early plasma cells.     

Telmisartan inhibits beta2-integrin MAC-1 expression in human T-lymphocytes

OBJECTIVE: The pathogenesis of atherosclerosis, a chronic inflammatory disease, is influenced by the renin-angiotensin system and especially by angiotensin II subtype 1 (AT1) receptor activation. Although pro-inflammatory properties of angiotensin II as well as anti-inflammatory effects of AT1 receptor antagonists are well known, the underlying mechanisms are poorly understood. METHOD AND RESULTS: In a prospective double-blind study, patients with hypertension and coronary artery disease were treated with either 40 mg telmisartan (n = 21) or placebo (n = 21) for 12 weeks. General markers of inflammation, such as high-sensitivity C-reactive protein (hs-CRP) and interleukin-6 (IL-6), and cell adhesion molecules, such as soluble intercellular adhesion molecule (s-ICAM-1) and the leucocyte adhesion molecule soluble-L-selectin (sL-selectin), as well as the lymphocytic expression of the beta2 integrin MAC-1, were assessed before and after treatment. Telmisartan therapy significantly decreased the lymphocyte beta2 integrin MAC-1 expression, whereas hs-CRP, IL-6, s-ICAM and sL-selectin remained unaltered. In-vitro experiments were conducted to clarify the mode of action. Cultured human lymphocytes were stimulated with either angiotensin II or phorbol-12-myristate-13-acetate (PMA)/ionomycin, alone or after pretreatment with telmisartan. Whereas angiotensin II exerted no effect on beta2-integrin MAC-1 expression in lymphocytes, telmisartan dose-dependently inhibited beta2-integrin expression in lymphocytes in the absence or presence of angiotensin II. CONCLUSION: The AT1 receptor antagonist telmisartan inhibits the expression of the pro-inflammatory beta2-integrin MAC-1 expression in lymphocytes independently of angiotensin II, suggesting an AT1 receptor-independent atheroprotective effect of this AT1 receptor antagonist.     

Trypanosoma cruz/-induced decrease in the level of interferon-7 receptor expression by resting and activated human blood lymphocytes

A substantial proportion of human peripheral blood mononuclear cells (PBMC) manifested a decreased capacity to express membrane interferon-γ receptors (IFN-γR) when co-cultured with Trypanosoma cruzi. Among the lymphocytes, B cells accounted for the bulk of this effect, evidenced by a marked drop in the proportion of CD19+ or CD20+ cells expressing IFN-γR. Decreased IFN-γR expression by B lymphocytes was seen as early as 3 h after co-culture with T. cruzi and persisted for at least 24 h. The parasite had no detectable effect on CD19, CD20 or DR antigen expression by B lymphocytes. Neither the proportion ofB cells expressing these markers nor the membrane density of these molecules varied significantly in the presence of T. cruzi. In PBMC cultures stimulated with Staphlyococcus aureus Cowan I (SACI), T. cruzi decreased the percentages of both IFN-γR+ and IFN-R +^bnght (cells expressing above-normal levels of surface IFN-γR) B lymphocytes. Cell-free filtrates of T. cruzi suspensions reproduced the suppressive effects of living parasites on IFN-γR expression by B cells. When T. cruzi was present, the intracellular levels of IFN-γR molecules in resting or SACI-activated B lymphocytes, represented by fluorescence intensity, were well below control values, suggesting that decreased surface expression resulted from suppressed IFN-γR synthesis. Among T (CD3+) cells, 10–8% to 39–6% (7 donors) expressed surface IFN-γR and did so at a very low level. These percentages were also reduced by T. cruzi. If occurring in the host, downregulated expression of IFN-γR could curtail the utilization of IFN-γ, known to play a critical role in host defence against T. cruzi infection.     

Status of major red cell blood group antigens on neutrophils, lymphocytes and monocytes

Previous investigators have reported that a number of red cell (RBC) antigens are found on neutrophils (PMN) and lymphocytes. However, there is a lack of consensus in the literature. Furthermore, few data are available concerning the occurrence of RBC antigens on monocytes. To address this problem, leucocyte fractions prepared from donors of known RBC antigen phenotype were analysed by fluorescence flow cytometry. Based on recent reports that PMN do not express ABH antigens and on the data presented here, the occurrence of major RBC antigens on leucocytes may be defined as follows: (1) lymphocytes express A, B. Lea and Leb antigens, depending on the ABH secretor (Se) status of the donor; (2) ABH and Lewis antigens cannot be detected on monocytes and PMN regardless of Se status; (3) D, E, e, C, c, Fya, Fyb, Fy5, Jka, Jkb, Jk, K, k, M, N, S, s, U, Vel, Coa, Lan, Jk3Yta, Dib, Ge, Sc:1 or Lub antigens were not detected on lymphocytes, monocytes and PMN; (4) lymphocytes, monocytes and PMN all express I, i, P and P1 antigens. The absence of selected Rhesus, Duffy, Kell, Kidd and other antigens on PMN is at variance with some previous reports. Furthermore, the distribution of RBC antigens on lymphocytes and monocytes has not been previously characterized using immunofluorescence flow cytometry.     

Cell-mediated lysis of lipid vesicles containing eye muscle protein: Implications regarding pathogenesis of Graves ophthalmopathy

We prepared artificial vesicles that are lysed upon cell-mediated immunological attack by human lymphocytes. These vesicles are made from a mixture of dimyristoyl lecithin, dipalmitoyl lecithin, and cholesterol, have eye muscle membrane protein (EMP) inserted into the bilayer wall, and contain intravesicular (99m)Tc marker. Injury to the vesicular membrane was assessed by measurement of (99m)Tc release. Thyroglobulin (Tg) and Tg-anti-Tg complex (TgA) bind to EMP-vesicles to an extent equal to or greater than to native eye muscle membranes in vitro; this binding requires the presence of normal human IgG. The role of Tg, TgA, IgG, and peripheral blood lymphocytes in altering membrane permeability was analyzed. Incubation of vesicles for up to 3 hr alone, with added IgG alone, or with further addition of Tg or TgA did not result in (99m)Tc release. Addition of lymphocytes from normal donors to the above four preparations showed release in the presence of TgA. Lymphocytes from each of eight patients with Graves ophthalmopathy caused release not only in the presence of TgA, but also in the presence of Tg. Separation of a patient's lymphocytes into high- and low-affinity rosette-formers (T and K cells, respectively) showed that cell-mediated vesicle lysis in the presence of TgA was greater with K cells than with T cells, while vesicle lysis in the presence of Tg was greater with T cells than with K cells. Vesicles made with inserted Tg but lacking EMP were not lysed by such T cells. Lymphocytes failed to induce permeability changes in vesicles containing other inserted proteins obtained from human nonextraocular muscle, liver, spleen, or adrenal, even if Tg or TgA were present. The results support the concept that muscle cell damage in Graves ophthalmopathy is immunological, cell-mediated, and of two types: (i) K lymphocytes reacting to immune complex, TgA, on the eye muscle cell surface (i.e., antibody-dependent cytotoxicity) and (ii) sensitized T lymphocytes reacting to Tg on the eye muscle cell surface. An antigenic role for EMP is possible, but has not been unequivocally proven.     

Stimulation of human lymphocyte proliferation and CD40 antigen expression by phosphorothioate oligonucleotides complementary to hepatitis B virus genome

SUMMARY. We have studied the proliferation and CD40 antigen expression of lymphocytes, and the cytotoxicity to monocytes, of antisense phosphorothioate oligodeoxynucleotides complementary to the SP II promoter of HBV mRNA (sequence I) and the X gene (sequence II) in patients with chronic hepatitis B. The oligo sequence I stimulated proliferation of both T and, to a lesser extent, B cells. The percentage of cells expressing CD40 in T and B cell co-cultures increased from 4.2% to 13.8% after oligo stimulation in patients, while it increased from 4.7% to 48.6% in healthy controls. The sense sequence (sequence III) of the X gene also enhanced the expression of CD40 antigen in patients with hepatitis B. The proportion of CD40 cells (26%) in a resting B-cell preparation from hepatitis B patients decreased to zero after a 5-day culture with sequence I, but IgG levels in the culture supernatant increased. The cytotoxic properties of monocytes were not influenced by the oligos. These findings indicate that antisense oligos against hepatitis B virus (HBV) have mitogenic effects on the proliferation of human lymphocytes in a non-specific manner and may activate T cells to express CD40 antigen.     

骨髓间充质干细胞对外周血T淋巴细胞亚群影响的体外实验研究

目的：探讨骨髓间充质干细胞（MSC）在体外对T淋巴细胞亚群及其分泌的细胞内因子的影响。方法：通过Ficoll—Hypaque梯度密度离心法分离出正常人骨髓单个核细胞，体外培养扩增MSC，获取第3代细胞。将其按照不同的比例加入双向混合淋巴细胞培养（MLC）体系中，在第1，3，5天，采用MTT比色法检测各组MLC中的T淋巴细胞的增殖情况，再用流式细胞术分析各组T细胞亚群及其内因子白介素（IL）-4和干扰素（IFN）-γ的分泌变化情况。结果：加入了MSC的MLC体系中，MSC对T淋巴细胞的增殖抑制具有剂量依赖性，且随着时间延长，抑制程度增强；其中，CD4^＋T细胞亚群受抑不如CD8^＋T细胞亚群显著；而根据T细胞内因子的变化，Th1和Tc1的比率较对照组有显著降低，而Th2和Tc2的比率却有轻度上升。结论：MSC在体外能够明显抑制T淋巴细胞的增殖，尤其是CD8^＋T细胞（CTL）。除此之外，它还能降低MLC体系中的Th1Tc1，升高Th2和Te2。所以在临床上可能有减轻移植后急性移植物抗宿主病（aGVHD）的发生，而保留移植物抗白血病（GVL）的潜力。     

Type I interferons directly regulate lymphocyte recirculation and cause transient blood lymphopenia

Early viral infection is often associated with lymphopenia, a transient reduction of blood lymphocyte counts long before the onset of clinical symptoms. We have investigated lymphopenia in mice infected with vesicular stomatitis virus (VSV) or treated with the Toll-like receptor (TLR) agonists poly(I:C) and R-848. In all cases analyzed, lymphopenia was critically dependent on type I interferon receptor (IFNAR) signaling. With the use of bone marrow-chimeric mice, radioresistant cells, such as stroma and endothelium, could be excluded as type I interferon (IFN-alpha/beta) targets for the induction of lymphopenia. Instead, adoptive transfer experiments and studies in conditionally gene-targeted mice with a B- or T-cell-specific IFNAR deletion demonstrated that IFN-alpha/beta exerted a direct effect on lymphocytes that was necessary and largely sufficient to induce lymphopenia. Furthermore, after treatment with R-848, we found that other cytokines such as TNF-alpha also played a role in T-cell lymphopenia. Investigation of the molecular mechanism revealed that lymphopenia was mainly independent of G protein-coupled receptors (GPCRs) and chemokines. In an adhesion assay, B cells of poly(I:C)-treated mice showed moderately increased adhesion to ICAM-1 but not to VCAM-1. In conclusion, our data identify a new effect of direct IFN-alpha/beta stimulation of lymphocytes that profoundly affects lymphocyte redistribution.