Preparation of monoclonal antibody to hepatocellular membranes and its application to induction of liver cell membrane damage

Monoclonal antibody (MoAb) to rat liver plasma membranes was prepared by hybridization of mouse immune lymphocytes with mouse myeloma cells, and was identified by the immunodiffusion method in a fraction of IgM secreted from the hybridoma thus obtained. In indirect immunofluorescence tests, specific fluorescence was detected only on the surface of rat hepatocytes, but neither on the cells from other organs of the rat nor on the hepatocytes of other species of animals, suggesting that the antibody may be organ- and species-specific. When the primary culture rat hepatocytes, labelled with isotopic chromium (51Cr), were treated with the MoAb together with complement, a specific release of 51Cr from the cells was found shortly after treatment, accompanied with bubbling of the cell membranes, and a significant release of 51Cr was observed at an MoAb concentration of 15 micrograms/ml or more. Without complement, or with inactivated complement, these reactions were not observed. These facts suggest strongly that the cell surface of the hepatocytes was damaged by the MoAb in the presence of complement.     

Selective Cytotoxicity of Two Rodent T Cell Lymphomas to Rat Yolk Sac Tumours Involves a Retroviral Envelope Protein Expressed by the Lymphoma

A Gross virus induced rat T cell lymphoma G1-Tc1 and a Moloney virus induced mouse T cell lymphoma YAC-1 are shown to exert a strong cytotoxic activity against rat yolk sac tumours but not to various types of rat, mouse or human normal cells or tumour cell lines including carcinomas, sarcomas, lymphomas and gliomas. Both lymphomas are CD3⁺, CD4⁻, CD8⁻ and T-cell receptor (TCR)αβ⁺. The cytotoxicity was not MHC restricted or dependent on the density of MHC class I of the target cells, and the mouse lymphoma killed the rat yolk sac tumour target. The cytotoxic action was fast and up to 80% specific killing was observed in 4-h ⁵¹Cr release assays. A rat B cell hybridoma was established from a Wistar/Furth (WF) rat immunized with the syngeneic lymphoma G1-Tc1 producing an immunoglobulin (Ig)G2c monoclonal antibody (MoAb) 1F2. This binds to the lymphomas G1-Tc1 and YAC-1 and also to a murine non-cytolytic Rauscher lymphoma RMA, but not to any other of several rat, mouse or human cell types tested. The 1F2 completely inhibited the killing of rat yolk sac tumours by the two cytolytic lymphomas, but did not interfere with the killing mediated by natural killer (NK) cells or cytolytic lymphokine-activated killer (LAK) cells. Immunochemical analysis of solubilized cell membranes of the lymphoma G1-Tc1 demonstrates that the 1F2 antibody recognizes an epitope on a retroviral gp 70 envelope protein. This indicates that a retroviral protein is involved in the lytic activity of the two lymphomas.     

Differential Cytotoxicity of Activated Lymphocytes on Allogeneic and Xenogeneic Target Cells

A competitive inhibition assay was used to define the specificity of the in vitro cytotoxicity of activated lymphocytes. Human and mouse effector cells were produced by prestimulation with phytohemagglutinin, pokeweed mitogen, or purified protein derivative for several days. The addition of increasing numbers of unlabeled Chang cells to a fixed number of stimulated human lymphocytes and ⁵¹Cr-labeled Chang cells gradually decreased chromium release. Admixture of unlabeled human bladder tumor cells, lung cells, or monkey kidney cells had a similar effect, whereas mouse L cells were not inhibitory. The reverse was found to be true when stimulated mouse lymphocytes and ⁵¹Cr-labeled mouse L cells were incubated. In this case, the addition of unlabeled L or rat tumor cells effectively inhibited ⁵¹Cr release, whereas human or monkey cells were less inhibitory. Both human and mouse cells inhibited the cytotoxicity of human effector lymphocytes towards mouse L cells. The cytotoxic effects of mouse lymphocytes on human cells were also decreased by addition of unlabeled human, monkey, or mouse cells, but in this case human cells were most inhibitory. The results indicate that activated lymphocytes recognize several surface structures on the target cells. Some of these structures may be shared by cells of different species origin, whereas others seem to be unique for cells from phylogenetically closely related species.     

Antibody-Mediated Cell-Dependent Immunity in Human Lymphocytes

A system is described which rapidly detects antibody-mediated cell-dependent immunity in multiparous women; their purified peripheral lymphocytes act as effector cells against ⁵¹Cr lymphocytes as targets, in the presence of specific antibody. Added complement was not required. The antibody can be detected in plasma, serum, and IgG preparations. The specific site of action of the antibody is primarily the target cell. Normal lymphocytes can be converted into effector cells. Antibodies demonstrated partial HL–A specificity and a high degree of sensitivity.     

Recognition of HLA-A1 by murine monoclonal antibodies

Abstract: Balb/c mice were immunized with cells from the mouse mastocytoma line P815 transfected with an HLA-A1 gene. The splenocytes of the immunized mice were fused with cells from the murine myeloma NS-1. In an initial screening, supernatants of growing cultures were tested for their binding capacity to the immunizing P815/A1⁺ cells as well as to P815/A2⁺ cells. Three out of 756 hybrids produced antibodies which bound to P815/A1⁺ cells only. They were cloned and further analyzed for their binding reactivity to reference B-lymphoblastoid cells from the Tenth International Histocompatibility Workshop. One monoclonal antibody, designated 6B11, reacted only with HLA-A1⁺ cells, while the two other antibodies, 3G3 and 7F10, appeared to detect antigenic determinants shared by HLA-A1, A3, A11, A26, and A30 (3G3) and by HLA-A1, A3, A11, A26, A28 and A30 (7F10). Flow cytometric studies on B-lymphoblastoid cell lines as well as on a series of tumor cell lines, including melanoma and colon carcinoma lines, confirmed the specificity of these antibodies. Monoclonal antibodies 7F10 and 6B11 were found to be of the IgM class and 3G3 of the IgG1 class. By complement-dependent ⁵¹Cr release experiments it was further shown that the two IgM antibodies 7F10 and 6B11 were able to lyse all cell lines of the HLA-A1 haplotype tested.     

The Effect of Alpha-fetoprotein on the Immune Response: Diminution of the Phagocytosis Capacity of Macrophages Cultured in vitro in the Presence of Mouse Amniotic Fluid or Alpha-fetoprotein

A study was carried out on the in vitro phagocytosis capacity of mouse peritoneal macrophages cultured in a medium supplemented with mouse amniotic fluid or alpha-fetoprotein purified from embryo extract. For this purpose ³H-TdR labelled staphylococci and ⁵¹Cr labelled fowl red blood cells were used. Both mouse amniotic fluid and alpha-fetoprotein inhibited phagocytosis, the antigen being incorporated in smaller amount than in the controls where the medium was supplemented with normal mouse serum.
In contrast to fetal glycoprotein, the presence of serum homologous proteins from adult animals did not inhibit the phagocytosis of macrophages but stimulated it.
The possible biological implications of this phenomenon are discussed.     

Alkali-Treated LPS of Yersinia enterocolitica does not Induce Expression of E-Selectin, ICAM-1 or VCAM-1 on Endothelial Cells but may Mediate Antibody- and Complement-Dependent Cell Injury

Lipopolysaccharide (LPS) prepared from a rough mutant of Salmonella typhimurium and deacylated enzymatically (dLPS) does not promote neutrophil adherence to human umbilical vein endothelial cells (HUVECs). This paper reports that similarly, a smooth form of LPS prepared from Yersinia enlerocolitica O:3, a serotype known to trigger reactive arthritis in humans, and treated with alkali (yersinia LPS-OH) failed to augment neutrophil adherence to HUVECs. Studies of the mechanism underlying the poor augmentation revealed that neither enzymatically deacylated LPS from Escherichia coli J5 (J5 dLPS) nor yersinia LPS-OH stimulated expression of endothelial cell adhesion molecules E-selectin, VCAM-1 and ICAM-1, whereas both Intact J5 LPS and yersinia LPS were stimulatory. Impaired up-regulation could not be explained by decreased binding of yersinia LPS-OH to HUVECs. Furthermore, ⁵¹Cr-labelled HUVECs treated with different concentrations of yersinia LPS-OH released ⁵¹Cr in the presence of anti-yersinia anti-0 antibody and complement. J5 dLPS and yersinia LPS-OH inhibited up-regulation of the adhesion molecules induced by J5 LPS and yersinia LPS but not that induced by tumour necrosis factor alpha.
Taken together, the results suggest that although yersinia LPS-OH can depress development of acute inflammation by inhibiting up-regulation of etidothelial-cell adhesion molecules, sufiicient LPS-OH is bound to induce cell injury and thereby inflammation in the prescence of specific antibody and complement. The findings may have pathogenetic implications in yersinia-triggered reactive arthritis characterized by dissemination of yersinia LPS throughout the body.     

The Generation of 'Cytotoxic' Macrophages in Mice during Infection with Influenza A or Sendai Virus

Injection of infectious but not of non-infections influenza A virus or of infectious or non-infectious Sendai virus intraperitoneally into mice induces the generation of plastic-adherent cells that arc able to effect release of ⁵¹Cr from labelled virus-infected target cells but not from labelled, uninfcctcd cells. Their activity is greatly diminished by exposure to silica or carrageenan but not by anti-Thy I antibody and complement treatment. Similarly, the activity of the cell preparation cannot be explained by contamination with natural killer or 'K' cells. Thus, the effector cells were identified as macrophages and for convenience are called 'cytotoxic macrophages'. The maximum cytotoxic activity was recovered from the peritoneal cavity 5 days after virus injection and declined thereafter. Although the effector cells are cross-reactive in that cells activated by an influenza A strain virus lyse target cells infected with the same or other A strain viruses or with Sendai virus, there is preferential lysis of cells infected with the homologous virus. The action of the effector cells is not H-2-restrictcd. Preliminary experiments showed that similar effector cells can be recovered from the lungs of mice 5 days after intranasal inoculation of infectious influenza virus, so they may contribute to the host control of the disease.     

Lipoxin A4 Induces Neutrophil-Dependent Cytotoxicity for Human Endothelial Cells

The authors have assessed the capacity of neutrophil granulocytes (PMN) to kill cultured human umbilical–vein endothelial cells (HUVEC) in vitro (as release of ⁵¹Cr) in response to the recently described double dioxygenation product of arachidonic acid, lipoxin A4 (LXA4). LXA4 conferred a marked cytotoxicity, whereas formyl–methionyl–leucyl–phenylalanine (fMLP) was less potent. The LXA4 and fMLP effects were dose dependent, with a maximum at 100 nM (which caused 2.7–and 2.3–fold increases of ⁵¹ Cr release, respectively, relative to buffer–treated controls). The LXA4 and fMLP responses increased with the PMN concentration, depended on the fetal calf serum concentration, incubation temperature and duration and the presence of calcium and magnesium ions.     

Antibody and Cell-Mediated Target Cell Lysis of Technetium-99m Labeled Erythrocytes

An assay for cell-mediated lysis of sheep erythrocytes has been developed using technetium-99m (^99mTc) as a radioisotopic label. Spleen cells obtained from sensitized BALB/c mice in combination with either non-immune or immune mouse anti-sheep RBC serum produced maximum release (100%) of ^99mTc. Non-immune spleen cells in combination with immune serum produced significantly greater release of ^99mTc compared to that observed with non-immune cells in the presence of non-immune serum. The kinetics of antibody-mediated complement dependent release of ^99mTc as a function of time and temperature paralleled the release of ⁵¹Cr and hemoglobin as an indicator of erythrocyte membrane injury. Because of its high specific activity, low spontaneous release, and ready availability in those institutions where diagnostic scanning is performed, ^99mTc appears to be particularly well suited for the assessment of both antibody and cell-mediated lysis of erythrocytes.     

Participation of the CD69 Antigen in the T-Cell Activation Process of Patients with Systemic Lupus Erythematosus

Accumulating evidence has implicated T cells in the pathogenesis of systemic lupus erythematosus (SLE). The CD69 antigen is an integral membrane protein rapidly induced on the surface of activated lymphocytes. We obtained CD4⁺ and CD8⁺ T cells from normal subjects and patients with SLE. The percentage of CD69 expression in freshly isolated cells and after in-vitro incubation with mitogens was quantified by three-colour immunofluorescent staining. Expression of this protein was increased in both CD4⁺ and CD8⁺ T-cell subsets from SLE patients when compared with normal cells, although the difference was significant only in the CD8⁺ T-cell subset ( P  = 0.05). Cellular activation increased CD69 expression. When stimulated with anti-CD2/CD2R or phytohaemagglutinin (PHA), the percentage and absolute numbers of CD69⁺ cells were lower in patients than in controls. Addition of anti-interleukin (IL)-10 monoclonal antibody (MoAb) increased the percentage of in-vitro CD69 expression in SLE cells. These results suggest that the peripheral blood lymphocytes from patients with SLE have an intrinsic defect that alters their activation process, including the expression of CD69, and might explain some of the T immunoregulatory abnormalities observed in these patients.     

The Effect of Capping by Anti-immunoglobulin Antibody on the Expression of Cell Surface Immunoglobulin and on Lymphocyte Activation

The ability of mouse spleen cells to bind anti-mouse immunoglobulin antibody labelled with ¹²⁵I (anti-mouse Ig Ab ¹²⁵I) in vitro was measured. The validity of this technique for quantifying the amount of surface immunoglobulin on mouse spleen cells was established by showing that the specific uptake of anti-mouse Ig Ab ¹²⁵I was linearly related to the number of spleen cells present. The technique was used to assess the exposed immunoglobulin determinants on mouse spleen cells after the redistribution of their surface associated immunoglobulins to a polar cap (capping) had been induced by anti-mouse Ig Ab. It was found that after capping the amount of surface immunoglobulin recovered almost to control levels, but showed no further increase. Treatment of spleen cells with chymotrypsin and papain but not trypsin markedly reduced the quantity of surface immunoglobulin. The rate of immunoglobulin resynthesis after chymotrypsinisation was not affected by previously capping the surface immunoglobulins. Concentrations of anti-mouse Ig Ab which induced capping of the surface immunoglobulin of spleen cells did not stimulate them to lake up (³H) thymidine although under the same culture conditions spleen cells were activated by phytohaemagglutinin and lipopolysaccharide. It is considered that capping is not in itself a sufficient stimulus to bring about in spleen cells either an increase in the density of the surface immunoglobulin or the initiation of DNA synthesis.     

Destruction of Sensitized Erythrocytes by Human Monocytes in Vitro: Effects of Cytochalasin B, Hydrocortisone and Colchicine

The purpose of this study was to characterize the destruction of sensitized erythrocytes by human blood monocytes in vitro The incubation in vitro of human monocytes with ⁵¹Cr-lalbelled human erythrocytes sensitized with IgG rhesus alloantibodies anti-D EAIgG anti-D) resulted in release of⁵¹Cr from the erythrocytes (lysis) as well as uptake of ⁵¹Cr-labelled erythrocytes by the monocytes (phagocytosis). The lysis of EAIgG anti-D by monocytes was not dependent on phagocytosis, because cytochalasin B, which inhibited phagocytosis of EAIgG, enhanced lysis. In contrast, hydrocorlisone and colchicine inhibited lysis, but had no effect on phagocytosis. These agents did not affect binding of EAIgG anti-D to monocytes. The effect of these agents on lysis corresponded to their effect on release of lysosomal enzymes by monocytes. The release of lysosomal enzymes, when induced by EAIgG anti-D, was, likewise, enhanced by cytoehalasin B and inhibited by hydrocortisone and colchicine. A significant correlation was found between lysosomal enzyme release and lysis. Together, these results strongly suggest that lysosomal enzymes. released by the monocytes when incubated with anti-D-sensitized erythroeytes, are responsible for the cytotoxic activity of these cells towards sensitized erythrocytes. The action of these enzymes only occurs over a short range, probably at the site of attachment of the erythrocyte, becuuse only erythrocytes that were bound to the monocytes were lysed. The finding of other investigators that removal of monocytes from suspensions of human mononuclear leucocytes results in a strong reduction in the cytotoxic activity of these leucocytes towards sensitized erythrocytes in vitro. was confirmed.     

Regulatory B cells as inhibitors of immune responses and inflammation

Summary: B cells positively regulate immune responses through antibody production and optimal CD4⁺ T-cell activation. However, a specific and functionally important subset of B cells can also negatively regulate immune responses in mouse autoimmunity and inflammation models. The lack or loss of regulatory B cells has been demonstrated by exacerbated symptoms in experimental autoimmune encephalitis, chronic colitis, contact hypersensitivity, collagen-induced arthritis, and non-obese diabetic mouse models. Accumulating evidence suggests that B cells exert their regulatory role through the production of interleukin-10 (IL-10) by either B-1, marginal zone (MZ), or transitional 2–MZ precursor B-cell subsets. We have recently found that IL-10-producing regulatory B cells predominantly localize within a rare CD1d^hiCD5⁺ B-cell subset that shares cell surface markers with both B-1 and MZ B cells. We have labeled this specific subset of regulatory B cells as B10 cells to highlight that these rare CD1d^hiCD5⁺ B cells only produce IL-10 and are responsible for most IL-10 production by B cells and to distinguish them from other regulatory B-cell subsets that may also exist. This review focuses on the recent progress in this field and the exciting opportunities for understanding how this unique B-cell subset influences diverse immune functions.     

The Serologic Detection of HL-A Antigens in Human Milk

The whey prepared from human milk has been shown to contain the HL-A antigens of the milk donor. The inhibition of specific and nonspecific anti-HL-A alloantisera by the antigens in whey was assessed by a microcytotoxicity assay employing ⁵¹Cr release. After centrifugation at 100,000 g for 1 h, significant serologic activity could be demonstrated by the antigens contained in both the insoluble fraction and the soluble fraction. The serologic activity of the HL-A antigens in whey was specific and dose-dependent. The HL-A antigens were not equally represented within the soluble fraction when compared to their distribution in the insoluble fraction, and this may indicate some antigen destruction upon solubilization.     

Anti CD5 Sustains the Proliferative Response of IgM-Activated Human CD5+B Cells

The CD5 molecule is expressed by most T cells but it is present on a minor B cell subset. Whilst several studies have provided information on the physiological role of T cell CD5, the functional role of CD5 on B lymphocytes remains unclear. To address this question, tonsillar CD5⁺ B cells were sorted by dual-colour fluorescence and FACS. Sorted cells were stimulated with polyclonal anti-IgM antibodies (Ab), and monoclonal (MoAb) F(ab')₂ fragments of anti-CD5. Proliferative responses were evaluated by enumeration of Ki-67 positive cells using quantitative flow cytometry. Co-stimulation with anti-CD5 MoAb for 3 days did not affect the anti-IgM and IL2-induced proliferation of CD5⁺ B cells. This was seen under conditions where the anti-CD5 was soluble, adsorbed to the microwells or cross-linked by anti-mouse antibodies. Fewer CD25⁺ cells were detected, however, in the presence of anti-CD5. In contrast, the proliferative response of CD5⁺ B cells prestimulated for 3 days with IL-2 and anti-IgM, was sustained in a further 3-day culture period when anti-CD5 was added. It is concluded that CD5 occupancy might provide an additional signal to activated CD5⁺ B cells favouring their proliferation and differentiation into autoantibody secreting cells.     

Anti-GD 3 Antibodies are Potent Activators of Human γ/δ and α/β Positive T Cells

The ganglioside GD3 has a variety of biological functions. These include stimulatory effects is on proliferation, natural killer activity and cytokine production by freshly isolated peripheral T cells. In this study we have characterized anti-GD3 antibody (MoAb Z21) mediated effects on T cell clones. Our data indicate that α/β TCR CD4⁺ and CD8⁺ as well as γ/δ TCR positive T cells can be stimulated resulting in proliferation and cytokine production. This effect could be blocked by cyclosporin A and did not involve the LFA-3 or CD4 molecule. Apart from IFN-γ and IL-2 production by T helper I and T helper 0 cells we have observed production of IL-4 and IL-10 by T helper 2 cells indicating that the GD3 molecule is not a marker for a certain functional T cell subset. In contrast to anti-CD3 mediated activation, the responsiveness of T cells to stimulation via GD3 was dependent on the cell surface expression of the molecule and could be enhanced by costimulation via CD2, CD3, CD26 or CD28. In addition, anti-GD3 antibodies delivered a potent costimulatory signal for antigen-induced proliferation of CD4⁺ T lymphocytes. In summary, our experiments illuminate the mechanisms of anti-GD3 antibody induced T cell activation.     

Development of Natural Killer Cell Activity and Genetic Resistance to Bone Marrow Transplantation with Age: Effect of Neonatal Thymectomy

The effect of neonatal thymectomy on the development of splenic and bone marrow natural cell-mediated cytotoxicity and on genetic resistance to bone marrow transplantation was examined in mice. Natural cytotoxicity was measured by a ⁵¹Cr release assay; the ability to engraft foreign bone marrow was assayed by the spleen colony method. The natural cytolytic response of spleen cells increased progressively from youth to early adulthood, whereas that of the bone marrow declined during the same age period. Neonatal thymectomy significantly elevated the natural killer cell response of young mice only (4 weeks, spleen; 6 weeks, bone marrow). In other experiments, neonatally thymectomized and sham-operated mice were lethally irradiated at 4 or 6 weeks of age and injected with 2.5, 5.0 or 10 million rat marrow cells. Six days later spleen colonies were markedly reduced in both 4- and 6-week-old neonatally thymectomized mice with all rat marrow cell doses tested. Neonatal thymectomy did not alter the percentage of erythroid versus other colonies at either 4 or 6 weeks. In both thymectomized and sham-operated mice the number of colonies increased with increases in marrow cell dose. The data are suggestive of a production and dissemination to the spleen of cells involved in the natural cytotoxic response from the bone marrow.     

Arming of Lymphoid Cells by IgG Antibodies Treated with Protein A from Staphylococcus aureus

Mouse spleen lymphocytes treated with rabbit IgG anti-sheep erythrocytes (SRBC) complexed with protein A of Stapbylococcus aureus (SpA) form rosettes with SRBC. The attachment of SRBC to lymphocytes was due to the binding of the SpA-IgG antibody complex to the surface of the lymphocytes and was thus considered 'arming' of the cells. Normal mouse spleen cells 'armed' with SpA-rabbit IgG anti-chicken erythrocytes (CRBC) kill specifically ⁵¹Cr-labeled CRBC 'in vitro' in the absence of free antibodies. The killing by these 'armed' cells is an effect of the cell-bound SpA-IgG antibody complex. Both the SRBC rosette formation and the cell-mediated CRBC killing was dependent on the concentration of the SpA-IgG antibody complexes used for 'arming' the cell. A 100-fold increase in rosette formation or in killing of target cells was recorded for lymphocytes treated with SpA-IgG antibody complexes in comparison with cell treated with noncomplexed IgG antibodies. The specific binding of SpA-IgG antibody complexes to the Fc receptors of mouse spleen cells was demonstrated by inhibition studies. More than 60% inhibition of the rosette formation and in the killing of target cells was shown for cell treated With normal rabbit IgG or its Fc fragment before addition of the SpA-IgG antibody complex.     

Size Distribution of Killer Cells During Allograft Response.

The generation of cytotoxic effector cells in the spleen and in the peritoneal cavity of C3H mice immunized with a single injection of H-2 incompatible P-815-X2 mastocytoma cells was studied. At different times after immunization the spleen cells were fractionated according to their size by one-g velocity sedimentation, and the fractions were tested separately for their cytotoxicity against ⁵¹Cr labeled P-8I5-X2 cells in vitro. The results indicate that the size of the killer cell changes during the immune response; the early cytotoxic effector cells are large cells (blasts) whereas the late effector cells are small cells (lymphocytes).