Increased Number of IgG Fc Receptors on Monocyte-Enriched Peripheral Blood Leucocytes from Patients with Rheumatoid Arthritis

The number of free Fc receptors (FcR) per cell and the association constant (K_ass) for the binding of monomeric IgG were determined for monocyte-enriched peripheral blood mononuclear cells, isolated from 16 patients with active classical rheumatoid arthritis (RA) and from 15 normal healthy donors. The assay system was based on binding under equili brium conditions of ¹²⁵I-labelled monomeric rabbit IgG to monocytes purified from peripheral blood on a continuous gradient of Petcoll. Monocytes from 14 untreated RA patients (6 seropositive, 8 seronegative) expressed on the average 4.8±1.3 × 10⁴ FcR/cell. This number was significantly higher (P<0.01) than that found in the control group (3.46±0.7 × 10⁴ FcR/cell). There was also a significant difference between the mean K _ass of the RA group and the control group-2.1±0.7 × 10³1/mol and 2.6±1.0 × 10³ 1/mol, respectively (0.05 >P> 0.01). Two seropositive RA patients receiving systemic treatment with penicillamine expressed the same number of FcR/cell as the mean of the control group (3.6 ± 10⁴). Levels of circulating immune complexes (CIC) and of the complement-factor C3 split product C3d were also measured. No correlation was found between the number of FcR/cell and the concentration of C3d, but there was a weak correlation between the number of FcR/ccll and the level of CIC.     

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.     

Formation of Hybrid IgM Subunits

A mixture of rabbit IgM anti-human A₁ red cell antibody and a human ¹²⁵I-labelled monoclonal IgM was reduced with mercaptoethanot and exposed to oxygen under conditions in which most of the half subunits coalesced to IgM subunits with reformation of interchain disulphide bonds. Hybrid molecules were demonstrated in the mixture by precipitin tests with autoradiography and by binding of ¹²⁵l to human A₁ red cells. By careful selection of IgM antibodies with high energy in each binding site, such hybrids may be useful 'IgM coats' for haemagglutination inhibition tests attempting to define sub-class-specific and genetically determined antigens on IgM.     

Decreased peripheral blood γδ T cells in patients with bronchial asthma

Many cell populations are thought to be involved in the etiopathogenesis of bronchial asthma. We examined by flow cytometry the relative and absolute number of CD3*, CD4*, CD8*γδ TcR* T cells. CD19* B cells; and CD56* natural killer (NK) cells in the peripheral blood of 26 adult patients with difficult-to-control asthma (DCA) and 22 patients with minimally symptomatic asthma (MSA). Statistically higher relative and absolute numbers of NK cells (18.39±10.67% and 0.38±0.17×10⁹/l) in comparison with healthy controls (ll.77±8.06% and 0.25±0.19×10⁹/l) and significantly decreased relative and absolute numbers of γδ T cells (3.02±2.16% and 0.06±0.04×10⁹/l) in comparison with controls (5.65+2.90% and 0.13±0.08×10⁹/l) in the DCA patient group were found. After pooling of data from both MSA and DCA patients and dividing the patients according to the presence of allergy, the relative and absolute numbers of 78 T celts were found to be diminished in both the allergy (3.77±2.98 and O.O7±0.O5 ×10⁹/l) and nonallergy (3.06±1.78% and 0.06±0.03 ×10⁹/l) groups in comparison with healthy controls. The reason for the low number of 78 T cells in the peripheral blood of patients suffering from bronchial asthma is under investigation.     

Real-Time Biospecific Interaction Analysis of a Natural Human Polyreactive Monoclonal IgM Antibody and its Fab and scFv Fragments with Several Antigens

Surface plasmon resonance (SPR) was used to investigate the kinetics of interactions between the human monoclonal polyreactive IgM antibody CB03, its Fab as well as its single-chain variable fragment (scFv) and different antigens. From these experiments apparent binding constants were determined and compared with binding constants obtained by ELISA experiments. In SPR studies with the complete antibody, the polyreactivity of the CB03 antibody as derived from ELISA experiments was confirmed.
Interaction of scFv with k casein and human myoglobin is strong evidence for the location of polyreactivity within the variable domains of the antibody.
Apparent binding constants of the complete antibody to immobilized K casein (9.2 × 10⁷ M⁻¹) and to human myoglobin (1.6 × 10⁷ M ⁻¹) are up to 83 times higher than those of Fab. The binding constants of the scFv to the above mentioned antigens are again about 10 times lower when compared with Fab, which is mainly due to the lower association rates of the complexes formed by the scFv.     

Copper-Catalysed Reoxidation of Human Monoclonal IgM

Human monoclonal IgM was dissociated into subunits by reduction with 0.01_M dithiothreitol and then separated from the latter with minimal loss of free sulphydryl groups by gel filtration. Incubation in pH 8.0 buffered saline at 25°C for 1 or 2 h resulted in oxidation of approximately 10% of the sulphydryl groups and no significant polymer formation. Incubation for 20 h resulted in oxidation of approximately 60% of the sulphydryl groups and appearance of 15–55% polymers sedimenting at 17S. Oxidation of the sulphydryl groups during the 20 h incubation period was decreased, and reassociation of the subunits was prevented by the addition of 10⁻³ M ethylenediamine tetraacetic acid to the buffered saline. In contrast, 90% of the sulphydryl groups were oxidized and 75% of the subunits were reassociated after only 30 min when incubated in 10⁻⁵ M cupric ion. The reassociated IgM sedimented as a major and a minor component at 15S and 19S, respectively. Urea-SDS polyacrylamide gel electrophoresis further demonstrated that extensive interchain bonding also occurred. Neither the yield of polymers nor the relative quantities of the reassociated components were altered by increasing the incubation time to 20 h.     

Binding Affinity of Human Autoantibodies: Studies of Cryoglobulin IgM Rheumatoid Factors and IgG Autoantibodies to Albumin

The binding affinity of cryoglobulin IgM rheumatoid factors (RF) for human IgG and of human IgG anti-albumin autoantibodies for HSA was measured by the molecular sieving technique. The binding affinities of the two autoantibodies were consistently low (10⁴-10⁵ I/M) as compared to the affinities of corresponding hyperimmune animal antibodies (10⁶-10⁸ I/M). The findings were discussed in relation to theories on human autoimmunity. The existence of strict autotolerance at the T cell level and of autoreactivity at the low affinity B cell level was considered to be best compatible with the findings of this study and with the major known facts of autoimmunity.     

Purification of Human Interferon by Antibody Affinity Chromatography, using Highly Absorbed Anti-interferon

Antibodies against partially purified human leucocyte interferon (PIF) were bound to Sepharose 4B and crude interferons applied on this affinity column were purified, up to 8 × 10⁵ interferon units (IFU) per mg protein in one step. Antibodies against PIF were absorbed with immobilized crude human leucocyte interferon bound to Sepharose, whereby antibodies against impurities were predominantly removed. Extensively absorbed antisera were coupled to Sepharose and used for antibody affinity chromatography of crude interferon preparations. Leucocyte and fibroblast interferons were purified in one step with around 100% recovery, up to 1 × 10⁸ IFU per mg protein, and Namalva interferon up to 2 × 10⁷ IFU/mg. SDS electrophoresis of affinity-purified leucocyte interferon revealed that the interferon activity appeared in two bands (19,000 and 23,000 D).     

The Interaction between Beta 2-Microglobulin (ßm) and Purified Class-I Major Histocompatibility (MHC) Antigen

The function of MHC class-I molecules is to sample peptides from the intracellular environment and present them to CD8⁺ cytotoxic T lymphocytes. To understand the molecular details of the assembly (and disassembly) of peptide-ß₂m-class-I complexes a biochemical peptidc-class-I binding assay has been generated recently and this paper reports on a similar assay for the interaction between ß₂m and class I. As a model system human ß₂m binding to mouse class I was used. The assay is strictly biochemical using purified reagents which interact in solution and complex formation is determined by size separation. It is specific and highly sensitive. The observed affinity of the interaction, K_D, is close to 0.4 nw. The rate of association at 37 C is very fasi (the k_a is around 5 × 10⁴/M/s) whereas the dissociation is slow (the k_d is around 8 × 10⁻⁶/s); the ratio of dissociation to association yields a calculated K_D close to the observed value. At 37° C almost all of the purified class I participates in binding of the exogenously offered ß₂m showing that a considerable exchange of the endogenous ß₂m occurs. Finally, it was demonstrated that exogenous ß₂m enhances binding to MHC class-I of short perfectly-matching peplides as well as longer peptides.     

Cytokine Dependence of Human to Mouse Graft-versus-Host Disease

Human peripheral blood leucocytes (PBL) induce chronic graft versus-host disease (GvHD) in non-conditioned severe combined immunodeficient mice. Chronic GvHD was observed in such animals after transplantation of 6 × 10⁷ human PBL per g body weight. However, acute xenogeneic GvHD results from grafting at least 2 × 10⁷ human PBL per g body weight to heavily conditioned murine hosts. The large numbers of human PBL were thought to be required fo produce above threshold amounts of certain cytokines. We show that treatment of the recipient mice with human interleukin 2 reduces the number of cells to inflict acute GvHD by a factor of ten. Human T cells and not B cells or macrophages, were previously shown to generate acute xenogeneic GvHD, when selected cell types from peripheral blood were grafted. Most of the infiltrating cells had the CD4⁺ phenotype. We demonstrate that CD4⁺ T cells are the main mediator, as the disease is abrogated by treating the mice with cytotoxic CD4 antibodies, but not with CD8 antibodies. A survival pattern, similar to that seen in GvHD, was induced by transplantation of a Herpesvirus saimiri transformed human CD4⁺ clonal T cell line in conjunction with daily interleukin 2 injections. Herpesvirus saimiri transformed human T cells allow easily reproducible graft properties in chimeric mouse models for human diseases.     

Kinetics of the Reaction between a Monoclonal IgM Anti-I and Rabbit Red Cells

Covalently linked subunits, subunits aggregated from two half subunits, half subunits, F(ab'μ)₂ and Fab'μ were prepared. K-values 1900–5800 times lower than those for intact IgM and rabbit red cells were found for the covalently linked subunits, the aggregated subunits, and F(ab'μ)₂ and 12,500–38,500 times lower for half subunits and Fab'μ. Only whole subunits and F(ab'μ)₂ agglutinated rabbit red cells; compared to intact IgM the titre was reduced markedly, but from the calculated amount of molecules bound per red cell, IgM was found to be on a weight basis only twice as effective as the subunits and F(ab'μ)₂ in agglutinating the cells.     

Primary Stimulation of CD4+ Cells in the Presence of IL-4 or IFN-γ Alters the Frequencies of Cytokine-Producing Cells at Restimulation

The induction of specific effector functions in naive T cells may be directed by accessory signals during activation. These could be elicited through binding to cell surface molecules or through factors secreted by antigen-presenting cells or other simultaneously activated cells. We have investigated the influence of CD8⁺ cells and of exogenousiy added cytokines (interleukin (IL)-2, IL-4 and interferon (IFN)-γ) on the cylokine production in splenic CD4⁺ T cells. IL-2, IL-4, IL-5 and IFN-γ production in CD4⁺ cells was measured at the single cell level during primary mitogen stimulation in vitro in the presence or absence of factors or CD8⁺ cells. On day 5 the cells were restimulated with mitogen alone and analysed to evaluate the short-term development of cytokine-producing cells in such cultures. Preactivation in the presence of either exogenous IL-4 or IFN-γ led to an increased production of IL-4 and IFN-γ respectively at restimtilation, and the effects of both IL-4 and IFN-γ were augmented by IL-2. After preactivation in the presence of IL-2 and IL-4, every third CD4⁺ cell could be induced to produce IL-4. Exogenous IL-4 or IFN-γ further decreased each other's production. Depletion of CD8⁺ cells before activation resulted in a slight increase of IL-4-producing cells, indicating that simultaneous activation of CD8⁺ cells will influence lymphokine production in CD4⁺ cells. The results suggest that the pattern of lymphokines induced in naive cells may be influenced by factors secreted by preactivated CD4⁺ and CD8⁺ cells, and that naive cells are preferentially 'recruited' to produce similar cytokines.     

Sequential Analysis of Distributions of Donor-derived Thymocytes Bearing T-cell Antigen Receptor (TCR) and Donor-derived la+ Cells in Thymuses of Fully Allogeneic Bone Marrow Chimera in Mice

Lethally irradiated SJL/J mice were reconstituted with B10 bone marrow cells, and the process of thymic reconstitution by donor derived cells positive for I- A or Vβ8 molecules was investigated. The donor-derived la⁺ cells appeared in the medulla on day 7 after reconstitution. The la+ cells became confluent up to day 14, and the cellularity in the medulla on day 17 was almost the same as that in the normal thymus. Dull Vβ8⁺ thymocytes were first recognized in the cortex on day 10 and were identifiable in the medulla by day 14. The Vβ8⁺ cells seemed to be mainly CD4⁺8⁺ double-positive. Furthermore, most of the Vg8'cells in the medulla of chimeras given cyclosporin A for 3 weeks after reconstitution appeared to be CD4⁺8⁺. The present findings demonstrate that CD4 8+ thymocytes which bear a low concentration of TCR exist in the thymic medulla at a relatively early stage when donor-derived la⁺ cells have already settled there. The coincidental appearance and coexistence of la⁺ cells and TCR⁺ thymocytes in the medulla suggest that these histological characteristics may be related to the selection of thymocytes in this area.     

Aspects of the Primed Lymphocyte Typing (PLT) Technique

The influence of different culture conditions in the primary and secondary cultures of the primed lymphocyte typing (PLT) technique was investigated with special reference to the discriminatory capacity of the PLT-cells generated. In the primary cultures, the maximal yield of PLT-cells was observed early (about day 7) and decreased thereafter, while the maximal specificity was obtained considerably later (about day 14).
In the secondary cultures, the optimal culture time was in the interval 42 h -72 h, and up to this culture length, γ-irradiation (2,200–8,800 rad) of the secondary stimulators had no effect on the ¹⁴C-thymidine uptake of the cultures. In U-form microtiterplates, the number of PLT-cells per well should not be less than 2.5×10⁴, and higher PLT-cell numbers (e.g. 5.0×10⁴ per well) may confer further robustness upon the technique. The PLT-cell response and the discrimination was only slightly influenced by the number of secondary stimulator cells in the interval 5×10⁴ to 2 × 10⁵ cells per well. Freezing of the PLT-cells under controlled conditions resulted in a minor loss of viable eosin-excluding cells, while the specificity of the PLT-cells was unaffected.
Even when the culture conditions are standardized, it is necessary to perform a normalization of the data in order to obtain reproducible results. The normalization procedure should include a compensation for the variation in (i) the general responding capacity of each PLT-cell and in (ii) the general stimulatory capacity of each secondary stimulator.     

Molecular assembly of RNA polymerase II from the fission yeast Schizosaccharomyces pombe: subunit–subunit contact network involving Rpb5

Background: Eukaryotic RNA polymerase II is composed of more than 10 polypeptide chains. The minimum and essential subunits for RNA synthesis have not yet been identified. Toward this ultimate goal, we analysed the topological arrangement of the putative subunits. Here we report a subunit–subunit contact network involving subunit 5 of the fission yeast Schizosaccharomyces pombe RNA polymerase II.
Results: The rpb5 ⁺ gene encoding subunit 5 of RNA polymerase II was cloned from the fission yeast Schizosaccharomyces pombe . The polypeptide predicted from DNA sequence of the rpb5 ⁺ gene consists of  210 amino acids with a calculated molecular weight of 23 914. The homology of the amino acid sequence is 55% and 43% with Saccharomyces cerevisiae RPB5 and human hRPB25, respectively. Far-Western blot analysis of S. pombe RNA polymerase II using ³²P-labelled recombinant Rpb5 fused to glutathione S-transferase (GST) as a probe, indicated that Rpb5 binds strongly to membrane-immobilized Rpb1, Rpb2 and Rpb3 and weakly to Rpb5 and a 15-kDa subunit (Rpb8 or Rpb11). In agreement with this result, the ³²P-labelled Rpb3 probe showed a strong binding signal against Rpb5 in addition to Rpb1 and Rpb2. The existence of Rpb5–Rpb3 contact was supported by detection of complexes formed between these two proteins synthesized in vitro using protein-immobilized beads.
Conclusion: Rpb3 and Rpb5, the putative subunits of RNA polymerase II, associate each other to form binary complexes. These two subunits also bind to the two large subunits, Rpb1 and Rpb2, independently.     

Sequential Antibody Affinity Chromatography of Human Leukocyte Interferon

Antibodies to crude leukocyte interferon (CIF) were bound to Sepharose 4B, and CIF was chromatographed. A relationship between recovered interferon and percentage binding of the antibodies to Sepharose 4B was observed. Highest recovery was obtained when only 50%-85% of the antibodies were bound When CIF was subjected to normal affinity chromatography, specific activity of 1–5 × 10⁶ interferon units (IFU) (69/19B-units)/mg protein was obtained. When this purified preparation was further chromatographed on a column mainly directed against normal cellular proteins and then concentrated by normal affinity chromatography, a tenfold increase in 'direct' specific activity was obtained. It is suggested that the 'indirect' specific activity is about 2 × 10⁸ IFU/mg protein.     

Binding of Helix Pomatia A Hemagglutinin to Human Erythrocytes and their Cells. Influence of Multivalent Interaction on Affinity

The binding of ¹²⁵I-labelled intact (hexavalent) and partially reduced (divalent) Helix pomatia A hemagglutinin to human A.₁, A.₂, A.₃, A₁B and A₂B erythrocytes, human lymphocytes, human lymphoblastoid and other tumor cell lines was investigated. The essential finding was that the association constants calculated for the interaction between hemagglutinin and A-erythrocytes were many orders of magnitude higher than the intrinsic association constant for the interaction between the hemagglutinin and the blood group A determinant. The latter value was 5–10³1/mole. The K-values for intact hemagglutinin and A and AB erythrocytes were in the order of 10¹⁰1/mole at 18–22 °C and pH 7.3. For partially reduced hemagglutinin the K-values were in the order of 5–10⁷ 1/mole. Multivalent interaction would seem to be the essential factor responsible for the high K-values in the cell binding experiments. Intact hemagglutinin reacted against A₁ and A₂ erythrocytes or against a human osteogenic sarcoma cell tine (2T) gave homogeneous binding curves in Scatchard's plot. Human lymphocytes and most lymphoblastoid cell lines lacked hemagglutinin receptors.     

Mitogenic Activation of Human Lymphocytes: a Protein A Plaque Assay Evaluation of Polyclonal B-Cell Activators

Using the protein A plaque assay, the capacity of various polyclonal B cell activators to induce differentiation in human B lymphocytes was investigated. Dextran sulphate and native Dextran were both virtually devoid of mitogenic properties, Lipopolysaccharide, however, was round to be a potent mitogen in human cells that, although giving rise to low DNA synthetic response, induced high numbers of immunoglobulin-synthesizing cells. Mean plaque-forming cell (PFC) numbers in healthy blood donors assayed on the optimal day (days 5–7) were 23, 493 IgM/10⁴ cells, 11, 288 IgG/10⁶ cells, and 2643 IgA/10⁶ cells. Values obtained in spleen cells, peaking at days 4–6, were slightly higher. Purified protein derivative (PPD) was equally or oven more-effective than lipopolysaccharide (LPS) in generating PFC of different subclasses in peripheral blood with mean of 29, 241 IgM/10⁶, 21, 269 IgG/10⁶, and 3681 IgA/10⁶. PPD furthermore induced a marked DNA synthetic response in human lymphocytes. These data suggest that LPS and PPD may both be used as functional markers in human cells when analysing patients with a suspected immunodeficiency state. It is suggested that cultures should be assayed using the protein A plaque assay, thereby being able not only to investigate the individual immunoglobulin classes but also to avoid the possible hazards involved in measuring antigen-specific responses in patients whose prior immunization to the antigen tested can never be totally excluded     

CD8+ Cells are the Main Producers of IL10 and IFNγ after Superantigen Stimulation

Purified CD8⁺ T cells were recently shown to produce TH1 as well as TH2 types of cytokines upon restimulation, indicating an important role for these cells in regulation of immune responses. However, it is not known if the CD8⁺ cells would contribute to cytokine production in the presence of cytokine secreting CD4⁺ cells. In the present study the authors have investigated the proportion of cytokine-producing CD4⁺ and CD8⁺ cells in the spleen after in vitro or in vivo stimulation. They found that stimulation of spleen cells with the superantigen Staphylococcal Enterotoxin B (SEB) in the presence of IL4 promoted production of IL10 and IFNγ predominately by CD8⁺ cells. In contrast, the production of IL4 was almost exclusively confined to the CD4⁺ subset. When priming with SEB in vivo before subsequent restimulation in vitro , a protocol previously shown to induce anergy, up to 80% of the IL10 and IFNγ positive cell expressed the CD8 marker. Taken together, these results emphasize the important role of cytokine-producing CD8⁺ cells and indicate that CD4⁺ and CD8⁺ T cells may, in a given situation, produce distinct cytokines.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.