Sayaka Utsumi

Co-operative interaction of subcomponents of the first component of complement with IgG: a functional defect of dimeric Facb from rabbit IgG

By following dissociation kinetics of radiolabelled C1q from rabbit IgG antibody-sensitized sheep red blood cells (SRBC) before and after its incorporation in the C1 complex, it was demonstrated that the binding stability is markedly enhanced by the presence of the C1r2-C1s2 subunit of C1 which by itself exhibits no significant binding capacity to immune complexes. The dissociation of C1q was decreased by up to 95%, the extent of decrease being pronounced as the cell surface IgG antibody density increased. However, such a stabilizing effect of C1r2-C1s2 was largely abolished when SRBC sensitized with the dimeric fragment F(acb)2 lacking C gamma 3 was used as the C1 binder, whereas the dissociation rate of uncomplexed C1q from F(acb)2-sensitized cells was similar to that from whole IgG-sensitized cells. It was also shown that, although the C1r2-C1s2 subunit is dissociated selectively from C1 bound to either IgG- or F(acb)2-sensitized cells in the presence of EDTA, it is held on much longer by the former cells than the latter cells. These results were taken to indicate that, although the C1 fixation by immune complexes of IgG is undertaken primarily by the interaction between C1q and the C gamma 2 domain, it is also strengthened by the secondary interaction between the C1r2-C1s2 subunit of C1 and the C gamma 3 domain or a structure which is dependent on the pair of C gamma 3 domains.

Regulation by glycophorin of complement activation via the alternative pathway

Abstract The effect of glycophorin on complement activation via the alternative pathway was examined by incorporating it into the liposome membrane with trinitrophenylaminocaproyldipalmitoylphosphatidylethanolamine (TNP-Cap-DPPE). Liposomes having incorporated TNP-Cap-DPPE onto the membrane activate the alternative complement pathway of guinea pig as reported previously, and the additional insertion of glycophorin was found to reduce their activating capacity on the alternative complement pathway. This inhibitory effect was cancelled by pretreatment of the glycophorin-containing liposomes with neuraminidase indicating that the sialic acid in glycophorin is playing a role in the regulation of alternative complement pathway-activation on the biological membrane.

Cytotoxicity of activated platelets to autologous red blood cells

Summary Gel‐filtered human platelets exerted lytic activity on autologous red blood cells (RBC) when they were co‐incubated at 37°C with platelet‐activating agents, such as thrombin, collagen, ADP. LPS or PMA in the absence of plasma. Lysis of activated platelets themselves did not occur during the incubation period examined. Morphological observations showed that RBC exposed to thrombin‐activated platelets were fragmented and/or transformed into spherocytes. This haemolytic reaction by thrombin‐activated platelets did not occur at 4°C, or in the presence of agents which inhibited glycolysis or elevated intracellular levels of cAMP, indicating that energy‐dependent and cAMP‐regulated platelet metabolism was required for this reaction. When platelets and RBC were incubated in the same vessel, but were prevented from coming into direct cell to cell contact by means of a membrane barrier, their cytotoxicity was reduced but not eliminated completely. No cytotoxic activity against RBC was detected in platelet‐free supernatants obtained by centrifugation after activation of platelets with thrombin. On the contrary, activated and washed platelets retained the activity. These observations suggested that the cytotoxic activity was carried by some diffusible and easily inactivated factors, which were continuously produced and liberated from activated platelets. Cyclo‐oxygenase inhibitors inhibited the haemolytic activity of thrombin‐activated platelets, suggesting a role for some products of platelet‐cyclooxygenase pathway in platelet‐mediated haemolysis. These results provide the first evidence for a direct role of activated platelets in mediation of RBC‐damage in the absence of any plasma factors.

Mechanisms of Neutralization of Endotoxin by Monoclonal Antibodies to O and R Determinants of Lipopolysaccharide

The protective potentials of antibodies to O and R core regions of lipopolysaccharide (LPS) have been amply substantiated (2, 7). However, the efficacy of antibodies to the toxic lipid A moiety itself is still ambiguous, and how antibodies to regions distal from lipid A can neutralize the toxicity remains to be clarified. We have compared the effects of mouse monoclonal antibodies (mAbs) of IgG class to these three regions of LPS on the biological activities as well as micellic structure of LPS, in order to shed light on the mechanism of neutralization of endotoxin by these antibodies.

Polymorphonuclear Leukocyte‐Inhibitory Factor of Bordetella pertussis

In vivo biologic effects of the polymorphonuclear leukocyte‐inhibitory factor (PIF) of Bordetella pertussis were tested by using two experimentally induced inflammatory processes in mice. The intravenous injection of a partially purified extract from phase I bacteria strongly inhibited the glycogen‐induced peritoneal infiltration of polymorphonuclear leukocytes (PMN) and the Arthus reactions, whereas little inhibitory activity was found in the extract from phase III bacteria. The activity was localized in the outer membrane of phase I bacteria, as was the in vitro PIF activity, and the two activities gave the same behavior in DEAE‐cellulose chromatography. Therefore the observed suppression of inflammatory processes in mice is probably due to the inhibitory action of PIF on the function of PMN in vivo.

Co-operation between the pair of Cγ2 domains in Clq-binding by rabbit IgG

The single site binding constants of rabbit IgG and its plasmin-derived fragments F(acb)2, Facb and F(ab)2 for human Clq were measured by the sedimentation velocity method. The intact IgG and F(acb)2 having the paired Cγ2 domains gave an identical association constant at 20°C (Ka) of 3.02 × 104M−1 in the presence of a physiological concn of salt and on the basis of six sites per Clq. The Clq-binding affinity was found to be decreased to 1.04 × 104M−1 in the reduced, monomerized fragment Facb. Under the same conditions F(ab)2, which is completely unable to activate the classical complement cascade, gave an apparent Clq-affinity of 0.36 × 104M−1. The results, together with previous observations, led us to the conclusion that the Clq-binding site of rabbit IgG is constituted associatively by the pair of Cγ2 domains, each of which providing a limited, complementary part of the binding free energy between IgG and Clq.

Preparation of membrane fraction from herpes simplex virus-infected cells which induce cytotoxic T lymphocytes.

The immunogenic capacity of herpes simplex virs (HSV)‐infected cells and their subcellular membrane fractions was investigated by assessing the anti‐HSV cytotoxic T lymphocyte (CTL) response in cultures of spleen lymphocytes from HSV‐primed BALB/c mice. Methylchloranthrane‐induced fibrosarcoma (Meth A) cells infected with HSV (HSV‐Meth A) were fixed either with glutaraldehyde or by heating at 56 C to preserve their immunogenic competence and then used as a stimulator. Microsomes and plasma membranes were prepared from HSV‐Meth A and their immunogenic activities were determined. Though the recovery of stimulatory activity in the plasma membrane fraction was half of that in the microsome fraction, the activity in the former was much more stable than in the latter and the plasma membrane fraction proved to be well qualified as an immunogen for anti‐HSV CTL induction. Upon purification, the specific activity of the membrane fraction, on the basis of protein concentration, increased 43‐fold.

Suppressive effect of IgG1 antibody on the complement activation of IgG2 antibody of the guinea pig

Abstract The effect of interposition of non-hemolytic antibody on the hemolytic activity of the complement-nxing antibody was examined. When mixtures of the hemolytic IgG2 and non-hemolylic IgG1 classes of guinea pig antibody to TNP ‡, the total amount being kept constant, were reacted with TNP-SRBC, the average number of complement-induced lesions per cell ( Z ) varied as a function of (proportion to IgG2) n . The experimental values for the exponent n fell in a range between 3 and 4. Comparison of the hemolylic activity of IgG2 in the mixture with IgGl ( Z i ) with that of the same amount of IgG2 without IgGl ( Z 0 ) gave a relationship: Z i = Z 0 × (proportion of IgG2) n − m , where m was obtained from the dose-response curve. Z 0 vs (concentration of IgG2) m . Since values for m obtained under the conditions used (1−2) were always smaller than those for n , the activity of IgG2 was apparently suppressed by the presence of IgG 1. From the C1-transfer experiments, the C1-binding step was shown to be affected. Results were tested by a simple hypothesis and the mechanism for the suppressive effect of IgG1 antibody was analysed.

Preparation of Stable Target Cells for Anti-Herpes Simplex Virus Cytotoxic T Lymphocytes

Using an avirulent strain of herpes simplex virus (HSV), SKa, and a methylcholanthrene induced sarcoma cell line, Meth A cells, we have developed a reliable target cell system for detection of cell‐mediated cytotoxicity directed against HSV‐infected cells. SKa‐infection in Meth A produced no progeny virus but induced HSV‐specific surface antigens as revealed by radioimmunoassay using 125I‐labeled HSV antibody. Spontaneous release of 51Cr from the SKa‐infected Meth A cells was no more than that from uninfected control cells but a strong spontaneous 51Cr release was produced in Meth A cells infected with KOS, a virulent strain which produced a progeny virus in Meth A and was lytic for the cells. When used as a target, SKa‐infected Meth A cells could detect HSV‐specific cytotoxicity by spleen and lymph node lymphocytes of mice immunized with SKa and KOS. This system also detected effector cytotoxic lymphocytes stimulated in vitro by mixed cultures of immune spleen cells and KOS‐infected Meth A cells. Thus, the system should be valuable in studies of cell‐mediated cytotoxicity directed against HSV‐infected cells.