Quantification of CD24 and CD45 antigens in parallel allows a precise determination of B-cell maturation stages: relevance for the study of B-cell neoplasias.

Quantitative expression, i.e. absolute number of monoclonal antibody molecules bound per cell, was evaluated for CD24 and CD45 by flow cytometry with standards of fluorescence intensity on a panel of normal and neoplastic B cells. The CD24 antigen was expressed at homogeneous high level in fetal bone marrow and liver. Its density decreased progressively in the other normal tissues in parallel with the B-cell maturation. The ratio between CD24 density measured on fetal bone marrow B cells and that seen on adult peripheral B cells was 6:1. The CD45 antigen density was lower on fetal bone marrow cells than in the more mature stages. Fetal spleen lymphocytes and all the mature B lymphocytes displayed the same CD45 density than that seen on normal adult peripheral T cells. The CD24/CD45 antigen density ratio was precisely related to the stage of B-cell maturation. The same pattern of variation of CD24 and CD45 antigen density was seen on B-cell neoplasias, with a significantly higher value of CD24 and lower value of CD45 in acute lymphoblastic leukemias than in chronic malignancies. CD24 and CD45 antigen levels were frequently out of the range observed in the corresponding normal population. Among ALL patients, a low CD24/CD45 antigen density ratio was associated with a good prognosis. These data confirm the interest of an absolute quantitative study for widely distributed antigens.

Reactivity pattern of a monoclonal antikeratin antibody (KL1).

A monoclonal antibody against keratins (KL1) from normal human stratum corneum was obtained using hybridoma techniques. Spleen cells from immunized BALB/c mice were fused with NS1, a mouse myeloma cell line, to produce hybrids. Antibody activity to epidermal keratins was tested using an indirect immunofluorescence test on cryostat sections of human epidermis and rabbit lip. A stable clone producing antikeratin antibody was isolated and an ascitic fluid was produced and used as a source of antibody (IgG1 kappa). KL1 was characterized by its immunohistochemical staining of various epithelia and by its recognition of 55-57 kilodalton (kd) keratin polypeptide from normal epidermis using the immunoblot technique. Frozen and deparaffinized sections of normal human epidermis, mucosa, and esophagus were stained by this antibody only in the upper cell layers as demonstrated by both indirect immunofluorescence and immunoperoxidase techniques. Approximatively 80% of normal keratinocytes isolated after trypsinization were labeled by KL1 whereas most negative cells showed basement membrane zone antigens. This confirmed differences in the expression of medium-sized polypeptides between basal and supra-basal cells during the course of human epidermal differentiation. All epithelial cells from other human epithelia (thymus, thyroid, bronchial mucosa, stomach, intestines) were positive with KL1 whereas nonepithelial cells and tissues did not show any staining. In view of these results KL1 promises to be a useful tool in the exploration of human epithelial differentiation.

Immunoassay for functional human soluble interleukin-6 receptor in plasma based on ligand/receptor interactions.

Soluble forms of most cytokine receptors, able to bind effectively to their respective ligands, have now been described. A soluble interleukin-6-binding molecule derived from the gp80 component of the multichain IL-6 receptor can be detected in biological fluids, and can act as an agonist of IL-6 activity. The clinical significance of the soluble receptor levels still remains to be explored. We took advantage of the characterization of an anti-IL-6 monoclonal antibody and of an anti-IL-6R monoclonal antibody that both bound to IL-6/IL-6R complexes to design an immunometric assay for the measurement of soluble IL-6R complexed to IL-6. This reaction scheme was designated as ELIA (enzyme-ligand immunoassay). When exogeneous IL-6 was added in excess to an sIL-6R containing sample, all sIL-6R was present in a complexed form. Thus, the reaction scheme could also be used to determine total sIL-6R concentrations. A recombinant sIL-6R standard was prepared from the supernatant of murine thymoma cells transfected with a gene coding for an extracellular portion of the IL-6 receptor. The assay permitted the precise and reproducible measurement of sIL-6R in serum or plasma. This approach is of general relevance for the determination of soluble cytokine receptors in biological fluids, provided that adequate anti-cytokine and anti-receptor antibodies are available.

Large scale and clinical grade purification of syndecan-1+ malignant plasma cells.

For cancer immunotherapy, it is usually necessary to obtain a large number of tumor cells from patients. We have previously reported that syndecan-I is present only on malignant plasma cells in samples from patients with multiple myelomatosis. We report here that this antigen is cleaved by chymopapain. This makes it possible to develop a rapid and clinical grade procedure to purify large numbers of myeloma cells using anti-syndecan-1 mAb, magnetic beads and chymopapain.

MLR-R and MLR-S gene products are expressed on different lymphoid cells.

The role of human B and T lymphocytes in the mixed lymphocyte reaction (MLR) was investigated using T- and B-enriched populations obtained either by centrifugation of E rosetting cells over a Ficoll-Isopaque gradient or after B or T cells had been killed by specific antihuman T lymphocyte antigen or antihuman B lymphocyte and monocyte antigen sera, respectively, in the presence of complement. Purified T lymphocytes responded to allogenic cells whereas they were unable to stimulate the MLR; in contrast, purified B lymphocytes were not activated but were found to be good stimulators. Treatment of the MLR, at the time of addition of tritiated thymidine, by antihuman T lymphocyte antigen serum and complement completely suppressed the thymidine incorporation, indicating that the bulk of the proliferative response was supported by the T lymphocytes. Additional experiments with populations depleted of complement or Fc receptor-bearing lymphocytes did not suggest that these B lymphocyte subpopulations played any major role in the MLR.

Heterogeneity of thymic epithelial cell (TEC) keratins--immunohistochemical and biochemical evidence for a subset of highly differentiated TEC in the mouse.

The mouse thymic epithelial network was studied using three different anti-keratin antibodies. One of these antibodies, KL1, exclusively recognized a small subset of medullary epithelial cells characterized by its content of a high molecular weight keratin (63 kD). Since epithelial differentiation is known to be associated with the acquisition of high molecular weight keratins, KL1-positive cells, which express the Ia antigen and secrete thymulin, may represent a subset of highly differentiated cells among mouse thymic epithelial cells (TEC). These data reflect the heterogeneity of the thymic epithelium and support the concept that distinct TEC subsets might provide the thymus with different microenvironments.

Phenotypic heterogeneity of B cell chronic lymphocytic leukaemia.

The peripheral blood lymphocytes from 39 patients from the Latvian S.S.R.T., U.S.S.R. with chronic lymphocytic leukaemia (CLL) have been phenotyped with various monoclonal antibodies representing the major clusters of differentiation (CD) used for phenotyping B cells. A clear delineation of two groups of patients was evidenced. The major group (33/39) possessed leukaemic cells bearing surface immunoglobulins (SIg) at a low density, Class II HLA, and CD5, CD24 and CD37 molecules but not CD21, CD22 and CD35. CD23 antigen was seen only once under microscope examination, but could be visualized by flow cytometry. CD6 antibody reacted with cells from about 1/3 of this group of patients. In the six patients of the second group the leukaemic phenotype was SIg+, Class II HLA+, CD5+, 24+, 37+, 21+, 22+, 35+, 23+ and 6-. The main finding is the concomitant expression of CD22, CD21 (CR2) and CD35 (CR1) molecules, all involved in B cell activation. It is not yet known whether these observations correlate with different clinical evolutions of the disease.

An anti-lymphocyte monoclonal antibody (HB8) which cross reacts with human dermal elastic fibers.

A monoclonal antibody (MoAb) was selected during a fusion aimed at the preparation of antibodies specific for human T lymphocytes. This antibody reacted against all lymphocytes, but was not specific for the lymphoid lineage because it also detected monocytes, granulocytes and platelets. On skin sections it was found to stain specifically the microfibrillar component of the elastic fibers. This observation illustrates the unexpected specificities often found when making MoAb and, although its significance is still unexplained, provides a unique tool having potential application for anatomy, histopathology, comparative biology and vascular pathology.

Subclustering of CD1 monoclonal antibodies based on the reactivity on human Langerhans cells.

Among the differentiation antigens expressed by lymphoid cells, CD1 monoclonal antibodies (MCA) distinguish a group of antigens expressed by cortical thymocytes. Some of them (OKT6 and BL6) have been shown to react with normal human skin Langerhans cells (LC). Using indirect immunofluorescence on skin sections and LC-enriched epidermal cell suspensions obtained by skin trypsinisation, we have studied the reactivity of eight new CD1 MCA. Our immunocytochemical observations indicate that four groups of CD1 MCA can be distinguished on the basis of their reactivity on LC. NA.1.84, SK9/Leu6, 10 D.12.2 and I19 identify LC in skin and in suspensions, but 4A.76 and NUT2 were negative in both. M.241 became negative after trypsinisation of epidermal cells and D47 reacted with an antigen expressed on LC only after enzymatic treatment. These results demonstrate the heterogeneity of the antigens recognized by CD1 MCA. Some of them do not react with normal human LC. Some antigens appear to be masked in the epidermis and are expressed only after trypsin treatment. Our data confirm the heterogeneity of CD1 molecules recently documented on the basis of biochemical analysis.

Subpopulations of circulating lymphocytes in adults with coeliac disease.

Circulating lymphocytes were enumerated in 25 patients with coeliac disease and in 24 healthy donors by immunofluorescent staining using monoclonal antibodies for T cell surface phenotypic markers T11 (mature), T4 (helper) and T8 (suppressor) or polyvalent antisera for surface immunoglobulins (B cells). Proportions of peripheral T and B cells and percentages and ratio of T cell subsets in coeliac disease were not significantly different from those in controls. Individual ratios of helper to suppressor T cells did not correlate with disease activity or with keeping the diet.

Generation of hybridoma antibodies to double-stranded DNA from non-autoimmune BALB/c strain: studies on anti-idiotype.

A hybridoma obtained between normal spleen cells from BALB/c mice (a non-autoimmune strain) and SP2-O-Ag 14 myeloma cell line was designated as HB2. These hybrid cells produced an IgM kappa-anti-ds-DNA antibody, but their specificity was limited to some polydeoxyribonucleotides such as natural ds-DNA from calf thymus, poly dG-poly dC, poly d(GC) and poly d(GC)-poly d(GC). In contrast, poly dA-poly dT, poly d(AT) were not recognized. The configuration of the nucleic acid helix plays a small role if any, in the building of the epitopes recognized by the hybridoma HB2 antibodies, while the presence of G and C appeared to be essential. These epitopes could not be found on ss- and ds-polyribonucleotides. B cells able to produce anti-ds-DNA antibodies are therefore present in non-autoimmune BALB/c mice, but not enough to produce the corresponding antibodies at a detectable level in the serum. Following immunization of BALB/c mice with hybridoma HB2 monoclonal antibodies, anti-idiotype antibodies were obtained which also recognized idiotopes present in the serum from both murine MRL/1 and human systemic lupus erythematosus (SLE).

In vivo migration of tonsil lymphocytes in rheumatoid synovial tissue engrafted in SCID mice: involvement of LFA-1.

Integrin-adressin binding is a critical step in lymphocte attachment to target tissues. The mucosal recognition systems (alpha E beta 7, alpha 4 beta 7, MADcam-1) have been implicated in the autoimmune process in rheumatoid arthritis. We developed a model for in vivo study of radio-labelled lymphocyte circulation and their attachment to human rheumatoid synovium. We studied the homing of tonsil lymphocytes, considered as mucosal lymphocytes, and the involvement of alpha E beta 7 integrin and LFA1 in the homing of tonsil lymphocytes. We engrafted human rheumatoid synovium subcutaneously in 6 week old SCID CB17 mice. Three weeks later, we injected intraperitoneally 20 IO6 human peripheral blood or tonsil mononuclear cells, previously labelled with 3 mCFi HMPAO-99mTc. A mouse total body scintigram was obtained 20 h postinjection. The same protocol was performed after treatment of the MNC and mAb against LFA-1 (CD11a) or alpha E beta 7 (CD103). Tonsil MNC retention in the rheumatoid synovial graft 20 h post-injection was enhanced compared to blood MNC (12731 +/- 8297cpm/200 pixel) versus 5982 +/- 4713cpm/200 pixel, p < 0.05). A monoclonal antibody against LFA 1 decreased the activity in the graft (4152 +/- 1287 cpm/200 pixel), p < 0.05. No significant difference in tonsil MNC attachment to rheumatoid synovial tissue was observed with a mAb against alpha E beta 7 (8057 +/- 5009 cpm/200 pixel). Our results showed an increase in radiolabelled mucosal MNC migration in synovial tissue engrafted in SCID mice compared with blood MNC. Moreover, the date suggest that LFA-1 but not the alpha E beta 7 integrin is involved in tonsil MNC binding to synovial tissue in RA.

Increase of class II HLA molecules on the membrane of B lymphocytes from patients with rheumatoid arthritis.

Using a novel cytofluorometric method of cellular antigen quantification, we examined peripheral blood mononuclear cells (PBMC) from patients suffering from rheumatoid arthritis (RA) for quantitative modification of class II human leucocyte antigen (HLA) molecules expressed on the surface. Class II HLA molecules were detected by indirect immunofluorescence with a monomorphic monoclonal antibody. No change was observed in the density of class II HLA molecules at the surface of monocytes of RA patients as compared to that of paired healthy subjects. We confirmed that the percentage of class II HLA-bearing T cells was slightly increased in RA patients versus controls, but the density of class II antigens per cell could not be determined accurately. An increase in the density of class II HLA molecules on RA B cells was shown, suggesting that a chronic activation stage of this population contributes to the disease.

The dendritic reticulum cell pattern in B cell lymphomas of the small cleaved, mixed, and large cell types: an immunohistochemical study of 48 cases.

An immunohistochemical study was designed to study the dendritic reticulum cell (DRC) patterns in 48 cases of B cell non-Hodgkin's lymphomas of the small cleaved, mixed, and large cell types, both follicular (20 cases) and diffuse (28 cases), in order to evaluate the possible influence of DRCs on homing and the differentiation of neoplastic B cells. Three DRC patterns were observed. In the follicular lymphomas, DRCs constituted nodular networks of variable density. In the diffuse lymphomas, DRCs were present either as isolated and scattered cells (17 cases) or constituted irregular meshworks of variable sizes (11 cases). These DRC patterns correlate with B cell immunophenotypes. Like follicular lymphomas, and unlike diffuse lymphomas without DRC networks, diffuse lymphomas with DRC networks constantly expressed the pan B antigens and one marker characteristic of normal germinal center cells, CD21 antigen, the C3d receptor. The finding of organized DRC networks in a significant number of diffuse lymphomas does not substantiate the hypothesis that DRCs may play a role in the homing of neoplastic B cells. The correlations observed between DRC patterns and B cell immunophenotypes suggest that the persistence and/or the development of DRC networks within follicular center cell-type lymphomas are related to the degree of functional differentiation of neoplastic B cells.

The Mi15 monoclonal antibody (anti-syndecan-1) is a reliable marker for quantifying plasma cells in paraffin-embedded bone marrow biopsy specimens.

Plasmocyte selective monoclonal antibodies (MAb) recognizing syndecan-1 have recently been described. They belong to a new cluster, CD138. Using the MAb MI15, we investigated the expression of syndecan-1 in routinely paraffin-embedded tissues. Nontumoral lymph nodes (25 cases) and bone marrow biopsy specimens (63 cases) showed strong membrane staining of plasma cells only, allowing accurate analysis of the nuclear structure. The MI15 positivity correlated with kappa and lambda light chain expression in the cytoplasm. The percentages of plasma cells calculated in bone marrow biopsy specimens after MI15 staining were, respectively, 2.1% (range, 1% to 4%) in normal bone marrows, 8.5% (range, 5 to 17) in reactive plasmocytosis, and 4.66% in monoclonal gammapathy of undetermined significance (MGUS) patients (range, 1 to 13), in the same range but slightly higher than those obtained on smears or on hematoxylin and eosin (H&E)-stained sections. In multiple myeloma (40 cases), all plasma cell types were marked, and Mi15 MAb gave additional information in 8 of 40 (20%) patients. In lymph nodes, Mi15 MAb reacted with Reed-Sternberg cells of classical Hodgkin's disease in 23 of 31 cases (74%) with variable intensity. In contrast, nodular lymphocyte predominance Hodgkin's disease (10 cases), most B cell lymphomas (88 of 107 cases) and all T cell lymphomas (30 cases) were negative. In B cell lymphomas, plasmocytomas (8 cases), plasmocytic lymphomas (2 cases), and 5 of 13 cases of immunoblastic lymphoma with plasmocytoid differentiation were stained. In lymphoplasmocytoid lymphomas (4 lymph nodes and 20 bone marrow biopsy specimens), only mature plasma cells were positive. Moreover, a wide distribution of syndecan-1 was observed in normal and tumoral epithelial tissues. Finally, Mi15 MAb appears to be a reliable marker for identifying and quantifying normal and tumoral plasma cells in paraffin-embedded bone marrow and lymph node samples.

Expression of the interleukin 6 receptor in primary renal cell carcinoma.

AIMS: Interleukin 6 (IL-6) is expressed in the majority of renal cell carcinomas and has an important role in the proliferation of some renal cell carcinoma cell lines. This action is mediated by two membrane proteins, gp80 (the IL-6 receptor; IL-6R), which binds IL-6, and gp130, which transduces the signal. The soluble form of gp80 (sIL-6R) is able to activate gp130 when complexed to the IL-6 molecule. These considerations prompted an investigation of IL-6R expression in this malignancy. IL-6, C reactive protein (CRP), and sIL-6R were also measured in serum and correlated to clinical and pathological features. METHODS: Immunostaining was performed on cryostat sections from renal cell carcinoma tumours with M91, an anti-IL-6R monoclonal antibody, using the alkaline phosphatase antialkaline phosphatase technique. The proliferation index was measured using the KI-67 monoclonal antibody. CRP, IL-6, and sIL-6R were measured in serum before nephrectomy, using an immunoenzymatic or immunoradiometric assay. RESULTS: There were significant differences in survival in patients with tumours larger than 8 cm, metastasis at diagnosis, high nuclear grade tumours, detectable serum concentrations of IL-6 (correlated to CRP serum concentration), more than 4% proliferating cells, and the presence of the IL-6R in situ. Furthermore, the serum IL-6 concentration correlated with tumour size and stage. The mean serum sIL-6R concentration was not significantly different from that observed in 40 normal subjects. Tumour IL-6R expression was present in 10 samples. There was a significant association between the presence of the IL-6 receptor in tumours and tumour stage, nuclear grade, proliferation index, and serum IL-6. CONCLUSIONS: This study revealed the importance of IL-6/CRP and IL-6R expression in situ as potential new prognostic factors and opens the way to new therapeutic strategies in renal cell carcinoma.

IL-6-induced changes in synthesis of alpha 1-acid glycoprotein in human hepatoma Hep3B cells are distinctively regulated by monoclonal antibodies directed against different epitopes of IL-6 receptor (gp80).

The synthesis of the human acute-phase alpha 1-acid glycoprotein (AGP) is primarily controlled by IL-6 and IL-1 in liver cells. In the present study, monoclonal antibodies against human gp80 interleukin-6 receptor (IL-6R) were utilized to study the role of the IL-6R in the control of the IL-6-induced AGP synthesis in the human hepatoma Hep3B cell line. Two of the 4 MAbs used in this study, M164 and M195, identified 2 different epitopes involved in IL-6 binding and two others, M91 and M182, recognized epitopes not involved in IL-6 binding. Dose-response experiments indicated that up to 55% of AGP synthesis was inhibited by 10(5) ng/ml of MAbs 164 or 195 when Hep3B cells were treated by IL-6 for 48h. Kinetics of the inhibition of AGP synthesis after addition of anti-IL-6R indicated that the decrease of the IL-6-induced AGP synthesis by Hep3B cells was obtained immediately after the addition of the anti-IL-6R MAbs. Of the two MAbs not involved in IL-6 binding, M91 was unable to interfere with the IL-6-induced AGP synthesis whereas, surprisingly, M182 decreased it by about 25%. Since M182 was also able to interfere with the proliferative response of an IL-6 dependent plasma cell line, our results suggested that M182 may be directed to a structure involved in the IL-6/IL-6R gp130 complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-6 receptor signaling. I. gp80 and gp130 receptor interaction in the absence of interleukin-6.

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. To study the physiological role of these soluble receptors, both proteins were purified from human plasma and the kinetic constants of equilibria between IL-6 and its natural soluble IL-6R (sIL-6R) and gp130 receptor (sgp130) were measured using surface plasmon resonance analysis. Unexpectedly, natural sIL-6R and natural sgp130 were found to interact (Kd = 2.8 nM) in the absence of IL-6. No interaction was seen between the recombinant soluble receptors or between either natural soluble receptor and its recombinant partner. This binary complex was not due to copurification of IL-6 and was detected in human plasma of healthy donors. It results from either direct interaction between the two natural soluble receptors or indirect binding mediated by a yet unidentified copurified plasma molecule playing the role of an IL-6 antagonist. Once formed, the binary complex was found to be unable to bind IL-6. Soluble gp130 had already been shown to inhibit IL-6 signaling by inactivating the IL-6/IL-6R complex. In addition we show that, in the absence of IL-6, circulating natural sgp130 is able to inhibit directly the circulating sIL-6R that is a strong synergic molecule of IL-6 signaling.

Tumor necrosis factor is a survival and proliferation factor for human myeloma cells.

Interleukin-6 (IL-6) is a major survival factor for malignant plasma cells. In patients with multiple myeloma (MM), cell lines whose survival and proliferation are dependent upon addition of exogenous IL-6 have been obtained. We show here that tumor necrosis factor-alpha (TNF-alpha) is also a survival factor for myeloma cell lines, although less potent than IL-6. The survival activity of TNF-alpha is not affected by anti-IL-6 or anti-gp130 monoclonal antibodies (mAbs). TNF-alpha also induces myeloma cells in the cell cycle and promotes the long-term growth of malignant plasma cell lines. As TNF-alpha is produced in patients with MM and associated with a poor prognosis, these results suggest that anti-TNF-alpha therapies could be useful in this disease.

Interleukin-6 receptor signaling. II. Bio-availability of interleukin-6 in serum.

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. With a view to studying the physiological role of these soluble receptors, both proteins were purified from human plasma. Surface plasmon resonance was used to measure the kinetic constants of equilibria between IL-6 and natural sIL-6R, and between the IL-6/sIL-6R complex and soluble gp130. Kd values were found to be 0. 9 and 2.3 nM respectively. Soluble natural IL-6R and gp130 were also found to interact with a Kd of 2.8 nM in the absence of IL-6. By using these Kd values, a mathematical simulation predicted that 1) within a large range of IL-6, sIL-6R and sgp130 concentrations, free IL-6 represents 30% of the total circulating cytokine, 2) sIL-6R overconcentrations lead to dramatic changes of the concentration of free IL-6, 3) increased concentrations of sgp130 should produce an efficient buffering effect on the IL-6/sIL-6R complex without incidence on the level of free IL-6. According to this model, the IL-6/sIL-6R complex appears to be an important support of IL-6 signaling in the most commonly encountered in vivo situations. The concentration of this complex is directly under the control of the concentration of sIL-6R; its bio-availability should be efficiently buffered by increased sgp130 concentrations.