Differential Expression of T-Cell Adhesion Molecules and LFA-1-Dependent Intercellular Adhesion in HgCl2-Induced Autoimmunity and Immune Suppression

Exposure of Brown Norway (BN) rats to HgCl₂ induces Th2-mediated systemic autoimmunity. In contrast, in Lewis rats, HgCl₂ induces immune suppression, mediated by CD8⁺ T cells. HgCl₂ was previously found to enhance expression of LFA-1, ICAM-1 and CD134 (OX40) on T cells in BN rats. In the present study, T cells from Lewis rats were studied at day 4 after injection of HgCl₂. CD8⁺ T lymphoblasts were significantly increased, which were predominantly CD45RC^hi, and which showed enhanced LFA-1 expression. Furthermore, CD4⁺CD45RC^hi T cells showed increased numbers of ICAM-1⁺ cells, whereas expression of CD134 and CD26 was relatively decreased in CD4⁺ T lymphoblasts. Ex vivo experiments demonstrated that HgCl₂- exposure of BN rats, but not of Lewis rats, significantly enhances PMA [phorbol 12-myristate 13-acetate]-induced lymphocyte aggregation, mediated by LFA-1 and ICAM-1. In conclusion, HgCl₂-injected Lewis rats show early signs of T-lymphocyte activation, predominantly on CD8⁺ cells. Strain-dependent effects of HgCl₂ on cell adhesion molecules and expression of CD134 may play an important role in development of either autoimmunity or immune suppression.     

TNF optimally activatives regulatory T cells by inducing TNF receptor superfamily members TNFR2, 4-1BB and OX40

TNF is a pleiotropic cytokine with intriguing biphasic pro-inflammatory and anti-inflammatory effects. Our previous studies demonstrated that TNF up-regulated FoxP3 expression and activated and expanded CD4+ FoxP3+ regulatory T cells (Tregs) via TNFR2. Furthermore, TNFR2-expressing Tregs exhibited maximal suppressive activity. In this study, we show that TNF, in concert with IL-2, preferentially up-regulated mRNA and surface expression of TNFR2, 4-1BB and OX40 on Tregs. Agonistic antibodies against 4-1BB and OX40 also induced the proliferation of suppressive Tregs. Thus, TNF amplifies its stimulatory effect on Tregs by inducing TNF receptor superfamily (TNFRSF) members. In addition, administration of neutralizing anti-TNF Ab blocked LPS-induced expansion of splenic Tregs and up-regulation of TNFR2, OX40 and 4-1BB receptors on Tregs in vivo, indicating that the expansion of Tregs expressing these co-stimulatory TNFRSF members in response to LPS is mediated by TNF. Altogether, our novel data indicate that TNF preferentially up-regulates TNFR2 on Tregs, and this is amplified by the stimulation of 4-1BB and OX40, resulting in the optimal activation of Tregs and augmented attenuation of excessive inflammatory responses.     

Lymphocyte migration into the CNS modelled in vitro: roles of LFA-1, ICAM-1 and VLA-4.

We examined the changes in intercellular adhesion molecule-1 (ICAM-1) expression on brain endothelium in response to tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). ICAM-1 is normally present on these cells and is induced over 24 hr by both cytokines with a time-course which matches enhancement in lymphocyte adhesion. Anti-lymphocyte function-associated antigen-1 (anti-LFA-1) (CD11a), anti-very late antigen-4 (anti-VLA-4) (CD49d) and anti-CD18 block binding of mitogen-activated lymphocytes to brain endothelium and the effects of anti-LFA-1 and anti-VLA-4 are additive. Anti-ICAM-1 does not however block adhesion, nor does depletion of endothelial ICAM-1 reduce lymphocyte binding. Titration of the interacting cells indicated that the antibody blocking is due to interference in the endothelial/lymphocyte interaction. None of the antibodies affect the binding of non-activated lymphocytes, which is itself normally much lower than that of activated cells. The time at which lymphocyte adhesiveness is greatest for the endothelium corresponds with the time at which the lymphocytes express highest levels of LFA-1 and VLA-4. The data show a role for LFA-1 and VLA-4 in the early interaction of activated lymphocytes with brain endothelium. Kinetic studies indicate that the ligand for VLA-4 is VCAM-1. The ligand for LFA-1 could not be determined with certainty, but if it is ICAM-1, the levels of ICAM-1 on brain endothelium are not critical.     

Remote T cell co-stimulation via LFA-1/ICAM-1 and CD2/LFA-3: demonstration with immobilized ligand/mAb and implication in monocyte-mediated co-stimulation.

Proliferative response of resting T cells generally requires not only cross-linking of the T cell receptor (TcR) but also co-stimulatory signals from accessory molecules. We here have used a "three-cell" model consisting of: (a) resting human CD4+ T cells as responders; (b) CD3 monoclonal antibody (mAb) OKT3 on latex beads as surrogate stimulators; (c) autologous monocytes as source of co-stimulation. As described by Kawakami et al. (J. Immunol. 1989, 142: 1818), T cell proliferation in this system is observed with paraformaldehyde-fixed monocytes if they have been activated and interleukin (IL) 1 beta/IL 6 is supplied. Since this three-cell system provides TcR cross-linking at a site spatially "remote" from co-stimulation, they help distinguish adhesion from signal transduction but the molecules that mediate co-stimulation in this system have not been identified. Our studies now demonstrate that co-stimulation by the monocytes is dependent on each of two receptor/ligand pathways CD2/LFA-3 and LFA-1/ICAM-1 since it is inhibited by each relevant mAb but not a variety of control mAb. The hypotheses that CD2 and LFA-1 could each mediate co-stimulation was tested in simplified model systems in which the monocyte was replaced with immobilized CD2 mAb or purified ICAM-1 presented on a separate surface from the CD3 mAb. The results in these simplified models demonstrate that on resting T cells either CD2 or LFA-1 molecules alone can mediate "remote" co-stimulation unlike most other T cell surface molecules. Co-stimulation requires IL 1 beta/IL6 both in the weaker LFA-1 ligand-mediated co-stimulation and at lower CD2 mAb concentrations in the stronger CD2 mAb-mediated co-stimulation. Thus: (a) the accessory cell function of stimulated fixed monocytes in T cell proliferation requires both the LFA-1/ICAM-1 and CD2/LFA-3 pathways; and (b) the T cell molecules CD2 and LFA-1 can give co-stimulatory signals that can act in a "remote" fashion.     

Lymphocytic Choriomeningitis Virus Infection is Associated with Long-Standing Perturbation of LFA-1 Expression on CD8+ T Cells

Flow cytometric analysis of splenocytes from mice infected with lymphocytic Choriomeningitis virus revealed marked and long-standing up-regulation of LFA-1 expression on CD8⁺, but not on CD4⁺ T cells. Appearance of CD8⁺ T cells with a changed expression of adhesion molecules reflected polyclonal activation and expansion which was demonstrated not to depend on CD4⁺ T cells or their products. Cell sorting experiments defined virus-specific CTL to be included in this population (LFA-1^hiMEL-14^lo), but since about 80% of splenic CD8⁺ T cells have a changed phenotype, extensive bystander activation must take place; this is indicated also by the finding that CD8⁺LFA-l^hi cells transiently express several markers of cellular activation, e. g. transferrin receptor, IL-2Rα and β. Analysis of cells from the cerebrospinal fluid of mice infected intracerebrally showed that virtually all T cells present belonged to the CD8LFA-lhi subset and, correspondingly, the ligand ICAM-1 was found to be up-regulated on endothelial cells in the inflamed meninges. Preincubation of LCMV-primed donor splenocytes with anti-LFA-1 markedly inhibited the transfer of virus-specific delayed-type hypersensitivity to naive recipients. Together, these findings indicate that up-regulation of LFA-1 expression is a critical factor involved in directing activated CD8⁺ T cells to sites of viral infection.     

Essential role of rho kinase in the ca2+ Sensitization of Prostaglandin F2α-Induced Contraction of Rabbit Aortae

Although the prostate gland is a rich source of α1-adreno- (α1-AR) and m1-cholino receptors (m1-AChR), the membrane processes associated with their activation in glandular epithelial cells is poorly understood. We used the whole-cell patch-clamp technique to show that the agonists of the respective receptors, phenylephrine (PHE) and carbachol (CCh), activate cationic membrane currents in lymph node carcinoma of the prostate (LNCaP) human prostate cancer epithelial cells, which are not dependent on the filling status of intracellular IP₃-sensitive Ca²⁺ stores, but directly gated by diacylglycerol (DAG), as evidenced by the ability of its membrane permeable analogue, OAG, to mimic the effects of the agonists. The underlying cationic channels are characterized by the weak field-strength Eisenman IV permeability sequence for monovalent cations ( P _K(25) > P _Cs(4.6) > P _Li(1.4) > P _Na(1.0)), and the following permeability sequence for divalent cations: P _Ca(1.0) > P _Mg(0.74) > P _Ba(0.6) > P _Sr(0.36) > P _Mn(0.3). They are 4.3 times more permeable to Ca²⁺ than Na⁺ and more sensitive to the inhibitor 2-APB than SK&F 96365. RT-PCR analysis shows that DAG-gated members of the transient receptor potential (TRP) channel family, including TRPC1 and TRPC3, are present in LNCaP cells. We conclude that, in prostate cancer epithelial cells, α1-ARs and m1-AChRs are functionally coupled to Ca²⁺-permeable DAG-gated cationic channels, for which TRPC1 and TRPC3 are the most likely candidates.     

Overexpression of CD82 on human T cells enhances LFA-1 / ICAM-1-mediated cell-cell adhesion: functional association between CD82 and LFA-1 in T cell activation

We previously demonstrated that CD82, expressed on both T cells and antigen-presenting cells (APC), plays an important role as a co-stimulatory molecule especially in the early phase of T cell activation. We also showed that the CD82 expression level is up-regulated on activated T cells and memory T cells. This up-regulation enhances both T cell-T cell and T cell-APC interactions. In this study, we further investigated the mechanism of CD82-mediated cell-cell adhesion. The enhanced adhesion between CD82-overexpressing Jurkat cells was completely blocked by anti-ICAM-1 / LFA-1 monoclonal antibodies. Increased interaction of LFA-1 with ICAM-1 was further confirmed by enhanced adhesion of CD82-overexpressing Jurkat cells to immobilized ICAM-1-Ig. CD82 co-immunoprecipitated with LFA-1 from Jurkat cells and CD82 and LFA-1 colocalized at an adhesion foci. These results suggest that the T cell stimulation via anti-CD3 cross-linking or phorbol myristate acetate treatment up-regulates CD82 expression, leading to the colocalization of CD82 and LFA-1, and results in enhanced interaction between LFA-1 and ICAM-1. In addition, a blocking experiment using monoclonal antibodies suggested that CD82 and LFA-1 molecules on APC are also important for the optimal activation of T cells. This is the first report that describes the enhancement of cell-cell interaction through LFA-1 and ICAM-1 by the overexpression of another cell surface molecule, CD82.     

自身免疫性甲状腺炎腺体内淋巴细胞Fas-L的表达及血清sAPO-1/Fas测定的意义

为观察自身免疫性甲状腺炎（ＨＤ） 腺体内淋巴细胞Ｆａｓ－ Ｌ的表达及血清ｓＡＰＯ－ １／Ｆａｓ的测定, 与ＨＤ病因及临床意义的关联。本文应用抗链霉菌抗生素蛋白－ 过氧化酶连接法（ＳＰ法）对甲状腺细胞学涂片进行淋巴细胞Ｆａｓ－ Ｌ 的表达的观察; 并应用ＥＬＩＳＡ 法测定血清ｓＡＰＯ－ １／Ｆａｓ。结果表明, ＨＤ 腺体内淋巴细胞Ｆａｓ－ Ｌ的表达显著高于ＧＤ 组和对照组, Ｐ＜ ０ ．００１ ;血清ｓＡＰＯ－ １／Ｆａｓ 水平,ＨＤ低于ＧＤ、对照组和正常组,Ｐ＞ ０ ．０５ 。上述结果提示,ＨＤ腺体内淋巴细胞Ｆａｓ－ Ｌ表达的观察, 有助于评估ＨＤ治疗方法, 对治疗结果的影响,并对ＨＤ的发展和预后进行判断。     

Lymphocyte adhesion and transendothelial migration in the central nervous system: the role of LFA-1, ICAM-1, VLA-4 and VCAM-1. off.

Lymphocyte adhesion to and migration across endothelial cell (EC) monolayers, derived from the rat blood-retinal barrier (BRB), were measured in vitro. The binding of concanavalin A (Con A)-activated peripheral lymph node lymphocytes and the migration of CD4+ T-cell lines could be significantly increased by treating the EC with interleukin-1 beta (IL-1 beta). To determine the role of various adhesion molecules during the processes of lymphocyte binding and transmonolayer migration (diapedesis), lymphocytes were treated with monoclonal antibody (mAb) specific for CD11a (alpha L subunit of leucocyte functional antigen-1; LFA-1), CD18 (beta 2 subunit of leucam family) and CD49d (alpha 4 subunit of very late activation antigen-4; VLA-4) and EC with mAb specific for CD54 (intercellular adhesion molecule-1; ICAM-1) and CD106 (vascular cell adhesion molecule-1; VCAM-1). Binding of the highly adhesive but non-migratory Con A-activated lymphocytes was inhibited by mAb to CD11a (reduced to 73% and 65% of control lymphocyte adhesion) and CD18 (42% and 54%) on non-activated and IL-1 beta-treated EC, respectively, but not by mAb to ICAM-1 or VCAM-1. Diapedesis of the highly migratory T-cell line lymphocytes was also blocked by antibodies to CD11a (reduced to 11% and 10% of control T-cell migration), CD18 (29% and 43%) but in addition was also inhibited by anti-ICAM-1 (17% and 53%) on non-activated and IL-1 beta treated EC, respectively. Both anti-VLA-4 and anti-VCAM-1 were also effective in producing a smaller reduction in migration, but only on IL-1 beta activated EC (66% and 58% of control migration, respectively). These studies indicate that lymphocyte adhesion to central nervous system (CNS) vascular EC is largely dependent on LFA-1 but not through its interaction with ICAM-1. In contrast, lymphocyte diapedesis is mostly supported through the LFA-1/ICAM-1 pairing, with a small proportion being mediated by VLA-4/VCAM-1 on IL-1 beta-activated EC. This latter pathway, however, also appears to be dependent on LFA-1 interacting with the EC.     

The role of ICAM-1/LFA-1 and VCAM-1/VLA-4 interactions on T helper 2 cytokine production by lung T cells of Toxocara canis-infected mice.

In order to study the effect of costimulatory signals on T helper type 2 (Th2) cytokine production, monoclonal antibodies (mAb) against cell adhesion molecules (CAM) were added to cells in culture obtained from the lungs of Toxocara canis (Tc)-infected mice followed by the determination of interleukin-5 (IL-5) and IL-4 in the supernatants of the culture. ES-stimulated IL-5 production in the supernatant of total lung cells was reduced by 25% when anti-intercellular adhesion molecule-1 (anti-ICAM-1) mAb, anti-CD11a mAb, or both anti-ICAM-1 and anti-CD11a mAb together were added to the culture. The addition of anti-CD18 mAb had no effects. Anti-vascular cell adhesion molecule-1 (anti-VCAM-1) mAb addition also reduced IL-5 production by 60%, although the addition of anti-very late activation antigen-4 (anti-VLA-4) mAb or both anti-VCAM-1 and anti-VLA-4 mAb together were less effective. In the case of anti-CD3 mAb stimulation, similar effects of mAb to CAM were observed. In contrast, IL-4 production induced by anti-CD3 mAb was reduced more markedly by the addition of either anti-ICAM-1 or anti-CD11a mAb than the combination of anti-VCAM-1 and anti-VLA-4 mAb. Similar effects of mAb to CAM were observed on the production of IL-5 and IL-4 by CD4+ T cells purified using a fluorescence-activated cell sorter. Coincubation with adherent cells was necessary for the significant production of IL-5 and IL-4 by CD4+ T cells. These results suggest that the VCAM-1/VLA-4 interaction is more important for IL-5 production by CD4+ T cells in the lungs of Tc-infected mice, and that the ICAM-1/lymphocyte function-associated antigen-1 interaction is more important for the production of IL-4.     

Receptor for α1-Microglobulin on T Lymphocytes: Inhibition of Antigen-Induced Interleukin-2 Production

The human plasma protein α₁-microglobulin (α₁m) was found to inhibit the antigen-induced interleukin-2 (IL-2) production of two different mouse T-helper cell hybridomas. α₁m isolated from human plasma and recombinant α₁m isolated from baculovirus-infected insect cell cultures had similar inhibitory effects. Flow cytometric analysis showed a binding of plasma and recombinant α₁m to the T-cell hybridomas as well as to a human T-cell line. Radiolabelled plasma and recombinant α₁m bound to the T-cell hybridomas in a saturable manner and the binding could be eliminated by trypsination of the cells. The affinity constants for the cell binding were calculated to be 0.4–1 × 10⁵  M ⁻¹ using Scatchard plotting, and the number of binding sites per cell was estimated to be 5 × 10⁵–1 × 10⁶. The cell-surface proteins of one of the T-cell hybridomas were radiolabelled, the cells lysed and α₁m-binding proteins isolated by affinity chromatography. SDS–PAGE and autoradiography analysis of the eluate revealed major bands with M _r-values around 70, 35 and 15 kDa. The results thus suggest that α₁m binds to a specific receptor on T cells and that the binding leads to inhibition of antigen-stimulated IL-2 production by T-helper cells.     

Role of CD8+ T Cells in Mercury-Induced Autoimmunity or Immunosuppression in the Rat

In Brown-Norway (BN) rats mercuric chloride induces an autoimmune disease characterized by an increase in serum IgE concentration, and by the production of anti-glomerular basement membrane antibodies responsible for a glomerulonephritis with a heavy proteinuria. (i) This disease results from a B-cell polyclonal activation probably due to frequent anti-class II T cells. (ii) The self limitation observed in this model is associated with both a decrease in the frequency of anti-class II T cells and the emergence of CD8+ T cells able to suppress these autoreactive T cells. (iii) In Lewis (LEW) rats which do not develop autoimmunity, HgC12 provokes the appearance of non-antigen-specific CD8+ T cells responsible for a depression of T-cell functions. The aim of this work was to test the effect of treatment with an anti-CD8 monoclonal antibody (MoAb) in both BN and LEW rates, Anti-CD8 MoAb-treated rats were effectively depleted in CD8+ T cells. However, neither the induction nor regulation phases of mercury-induced autoimmunity were modified in BN rats. Mercury-induced immunosuppression in LEW rats was abrogated; however, depletion in CD8+ T cells did not allow the disease to occur in that strain. Finally, CD8 depletion induced in normal BN rats rats the appearance of rare anti-class II T cells showing that these cells are normally present in that strain but negatively controlled by suppressor T cells.     

Family studies with the chromosome 9 markers ABO, AK1, ACONs and 9qh

We have failed to measure the recombination fraction between 9qh and the ABO:AK1 linkage group. Since the total male map distance along chromosome 9 is likely to be at least 120 cM, calculated from male chiasmata counts (Hultén, 1974) they may well not be within measurable distance of each other. ACONs does not lie between ABO and AK1, but there is so little information on this marker from family studies that it could lie almost anywhere else on chromosome 9 and might be within measurable distance of 9qh. The map of chromosome 9 based on studies on families with normal karyotypes is summarized in Fig. 1. We hope to improve this map in a subsequent paper based on observations on families with abnormal karyotypes.     

Different requirements of ICAM-1/LFA-1 adhesion in allorecognition and self-restricted antigen recognition by class II-specific T cell clones

We have analyzed the influence of non-antigen-specific interactions between ICAM-1 and LFA-1 in target recognition by allospecific and antigen-specific Tcells at the clonal level, using human and mouse fibroblasts transfected with HLA-DR1 or DR2 with or without co-expression of ICAM-1, as antigen-presenting cells. The results show a great heterogeneity in the requirements for ICAM-1/LFA-1 interactions for antigen-specific and alloreactive T cell responses and this requirement may depend on the avidity of any particular interaction. The data also show that for most alloreactive clones, ICAM-1/LFA-1 adhesion is not sufficient to facilitate efficient T cell recognition of its target molecule. HLA class II recognition by a large proportion of the DR1- and DR2-specific alloreactive clones studied was different for class II molecules expressed on murine or human fibroblasts compared to human lymphoid cells, and was independent of ICAM-1 expression on the stimulator cells. The inability of some T cell clones to recognize HLA-class II expressed on non-lymphoid cells suggests the absence of specific epitopes and could be due to the lack of the relevant peptides, either because they are derived from species-specific proteins or to differences in processing of endogenous antigen in the transfected stimulator cells.     

Antisense Oligodeoxynucleotides for IL-2, c-myc and Transferrin Receptor Synchronize Mitogen-Activated Lymphocytes in the G1 Phase

In order to clarify the role and interrelationship of c- myc , interleukin-2 (IL-2), and transferrin receptor (TfR) expressions on PHA-stimulated lymphocyte prohferation, we examined the effects of antisense oligodeoxynucleotides directed against c- myc , TfR, and IL-2 mRNAs on DNA synthesis and cell-cycle phase. Exposure of PHA-stimulated lymphocytes to each antisense oligomer resulted in approximately 75 80% inhibition of DNA synthesis. TfR expression was not inhibited in PHA-stimulated lymphocytes by c- myc or IL-2 antisense oligonucleotides, suggesting that the expression of c- myc , TfR, and of IL-2 is regulated by an independent mechanism. All three antisense oligonucleotides for c- myc , TfR and IL-2 synchronized mitogen-activated lymphocytes to the G₁ phase as assessed by morphologic blast formation and cell-cycle phase analysis.     

Linkage relationships between the three phosphoglucomutase loci PGM1, PGM2 and PGM3

1. Phosphoglucomutase phenotypes have been studied in several generations of the family of an individual heterozygous at each of the three loci, PGM₁, PGM₂, and PGM₃. 2. PGM₁ and PGM₂ phenotypes were determined using red cells. Fibroblasts grown in tissue culture were used for PGM₃ phenotyping. 3. The family results support the genetical hypothesis based on the analysis of dizygotic twin pairs that the PGM₃ isozyme patterns found in the placenta are determined by two alleles, PGM¹₃ and PGM²₃. 4. Locus PGM₃ is not closely linked to locus PGM₂ 5. The data also support the previous findings that locus PGM₁ is not closely linked to PGM₂ or PGM₃.     

Adhesion molecules from the LFA-1/ICAM-1, 3 and VLA-4/VCAM-1 pathways on T lymphocytes and vascular endothelium in Graves' and Hashimoto's thyroid glands

Lymphocytic infiltration of the thyroid gland in autoimmune thyroid disorders requires, as a first step, their attachment to endothelial cells (EC) and, subsequently, their interaction with thyrocytes and extracellular matrix proteins. A number of different ligand molecules have been identified to mediate the interaction between EC and leukocyte subpopulations. In this study, we examined by flow cytometry and immunohistochemical techniques, the expression of integrin receptors and their counter-receptors by infiltrating lymphocytes and vascular endothelium in thyroid glands from patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT). A high proportion of GD intrathyroidal T lymphocytes expressed the CD69 and gp95/85 (Ea2) activation antigens as well as an increased number of LFA-α_L, VLA-α1, -α4, -α5, and -β1 integrin receptors, as compared with peripheral blood T lymphocytes from the same patients. The expression of intercellular adhesion molecule (ICAM)-1 was increased in EC from GD and HT thyroids. In addition, an up-regulated de novo expression of vascular cell adhesion molecule (VCAM)-1 was found in EC in GD and HT thyroids, with no reactivity in control thyroids. Dendritic cells in thyroid lymphoid follicles were also positive for ICAM-1 and VCAM-1. In addition, most of intrathyroidal mononuclear cells expressed the ICAM-3 adhesion molecule. This enhanced expression of ICAM-1 and VCAM-1 by thyroid EC in GD and HT may reflect their ability to regulate leukocyte trafficking and activation by means of the expression of specific ligand molecules. Our data suggest that both the LFA-1/ICAM-1, ICAM-3 and VLA-4/VCAM-1 pathways could play a relevant role in localizing and perpetuating the autoimmune response in the thyroid gland in autoimmune thyroid disorders.