Antibody response of mice to lactate dehydrogenase-elevating virus during infection and immunization with inactivated virus

BALB/c and Swiss mice were infected with lactate dehydrogenase-elevating virus (LDV) or immunized with glutaraldehyde-inactivated or ether-extracted virus and their plasma was monitored for anti-LDV IgG and IgM levels by ELISA and indirect fluorescent antibody staining, for neutralizing antibodies, for sensitized antibody-virus complexes, for immune complexes, and for total plasma IgG and IgM. In infected mice, anti-LDV IgM was transiently formed during the first 2 weeks post infection (p.i.) but only at a low level. Anti-LDV IgG was produced in a biphasic manner with an initial peak at about 10 days p.i. and a secondary rise reaching a maximum level 30-80 days p.i. which was retained throughout the persistent phase of infection. The concomitant appearance of comparable levels of low molecular weight immune complexes suggests that most anti-LDV IgG was complexed with LDV proteins. Also, as early as 10 days p.i., infectious antibody-LDV complexes developed, which were neutralizable by rabbit anti-mouse IgG, whereas antibodies that neutralize the infectivity of exogenously added LDV appeared only 1-2 months p.i. Throughout infection, most of the anti-LDV IgG was directed to VP-3, the envelope glycoprotein of LDV, which was found to exist in at least 10 distinct forms ranging in molecular weight from 24 to 42 kDa. Anti-LDV IgG levels as high as those observed in infected mice developed in mice immunized with inactivated LDV. Antibodies to glutaraldehyde-inactivated LDV were also mainly directed to VP-3, but exhibited no neutralizing activity. The polyclonal B cell activation associated with a persistent LDV infection and the formation of immune complexes were not observed in mice immunized with inactivated virus.     

Regulation of immune complexes during infection of mice with lactate dehydrogenase-elevating virus: studies with interferon-gamma gene knockout and tolerant mice.

Mice persistently infected with lactate dehydrogenase-elevating virus (LDV) develop circulating IgG-containing hydrophobic immune complexes, with a molecular mass of 150 to 300 kd, which bind to the surfaces of high-capacity enzyme-linked immunosorbent assay (ELISA) plates. LDV infection also stimulates polyclonal B-cell activation and autoimmunity. For this study, interferon-gamma gene knockout (GKO) mice were utilized to study circulating immune complexes and other parameters of LDV infection. The kinetics of LDV viremia, formation of plasma IgG anti-LDV antibodies, and LDV replication in the spleen and liver were essentially normal in GKO mice. Polyclonal activation of B cells, as reflected by increased total plasma IgG concentration during LDV infection, was found to be intact in GKO mice, although at a lower magnitude than in control mice. The plasma concentration of IgG-containing hydrophobic immune complexes was reduced about 75% in LDV-infected GKO mice relative to normal LDV-infected controls. Allogeneic tissue responses were also found to be reduced in LDV-infected GKO mice relative to those in normal LDV-infected controls. These results dissociate specific anti-LDV immunity from formation of hydrophobic immune complexes, show that the IgG anti-LDV response as well as LDV replication in the spleen and liver are insensitive to physiological levels of interferon (IFN)-gamma, and suggest that IgG-containing immune complexes stimulated by LDV infection are a marker for autoimmunity.     

Regulation of maternal-fetal virus transmission in immunologically reconstituted SCID mice infected with lactate dehydrogenase-elevating virus.

Immunodeficient SCID (C.B-17 scid/scid) mice with persistent lactate dehydrogenase-elevating virus (LDV) infection failed to produce IgG anti-LDV antibodies, and during chronic infection transmitted virus infection to 95% of their offspring. In contrast, normal mice infected 15 or more days prior to giving birth produced IgG anti-LDV antibodies and transmitted LDV infection to only 0-46% of their fetuses. Transplacental transmission of LDV infection was dependent on the timing of maternal infection. Adoptive transfer of immune competence to LDV-infected SCID mice resulted in fetal protection from maternally transmitted virus infection. Fetal protection correlated with the presence of maternal IgG anti-LDV but not with fetal levels of IgG anti-LDV, and the levels of viremia in nonimmune SCID mice did not affect transplacental virus transmission. These results demonstrate the importance of maternal immunity in protecting the fetus from infection, and validate the use of this mouse model for investigation of immune mechanisms of transplacental virus transmission.     

Immune response to lactate dehydrogenase-elevating virus: isolation of infectious virus-immunoglobulin G complexes and quantitation of specific antiviral immunoglobulin G response in wild-type and nude mice.

Lactate dehydrogenase-elevating virus (LDV) causes a normally benign persistent infection of mice, resulting in a life-long viremia characterized by the presence of circulating infectious immune complexes, impaired clearance of certain enzymes from the blood, and modification of the host immune response to various heterologous antigens. In this study, we isolated infectious immunoglobulin G (IgG)-LDV complexes in the plasma of persistently infected mice by adsorption to and elution from protein A-Sepharose CL-4B. We found that practically all infectious LDV in the plasma of persistently infected mice is complexed to IgG. LDV infectivity in these complexes was partially neutralized, but could be reactivated by treatment with 2-mercaptoethanol. We also quantitated total plasma IgG and anti-LDV IgG in wild-type and nude Swiss and BALB/c mice as a function of the time after infection with LDV by radial immunodiffusion and an enzyme-linked immunosorbent assay, respectively. Total plasma IgG levels nearly doubled in BALB/c mice during 150 days of infection. IgG levels in uninfected nude mice were only 20% of those in uninfected BALB/c mice, but during infection with LDV increased to approximately those found in uninfected BALB/c mice. Anti-LDV IgG levels were almost as high in nude mice as in normal BALB/c mice. Isoelectric focusing of purified IgG from BALB/c mice showed that LDV infection resulted in the enhanced synthesis of all 16 normal IgG fractions that we could separate by this method, which suggests that LDV infection results in polyclonal activation of IgG-producing lymphocytes.     

Persistent infection of mice by lactate dehydrogenase-elevating virus: effects of immunosuppression on virus replication and antiviral immune responses

Maximum plasma titers (10(9)-10(10) ID50/ml) of lactate dehydrogenase-elevating virus (LDV) in mice are observed one day after infection, but then decrease 4-5 log during the next 5 weeks to attain a persistent steady-state level for the remainder of the life of the animal. The decrease in plasma LDV level during the first 5 weeks after infection and long-term viremia were not affected by lethal X-irradiation of the mice, daily injections of cyclosporin A or depletion of the mice of T cells by treatment with anti-CD4, anti-CD8, or anti-Thy1.2 monoclonal antibodies, although these treatments inhibited the formation of anti-LDV antibodies. LDV viremia was also the same in nu/nu and nu/+ Swiss mice, though the former did not mount an anti-LDV immune response, while the latter did. The appearance of anti-LDV neutralizing antibodies in infected mice 1-2 months after infection or the injection of infected mice with high doses of anti-LDV neutralizing monoclonal antibodies also did not affect the level of LDV viremia. Repeated treatments of infected mice with either cyclophosphamide or dexamethasone caused 1-2 log increases in plasma LDV titers. Although cyclophosphamide treatment prevented the formation of anti-LDV antibodies, dexamethasone caused an increase in plasma LDV levels without affecting anti-LDV antibody formation. We conclude that an anti-LDV immune response does not play a significant role in controlling LDV replication in mice. The data support the view that within 1 day after infection of a mouse, all LDV-permissive macrophages, which appear to be the only cells supporting LDV replication in the mouse, are destroyed as a result of a cytocidal infection by LDV. Subsequently, LDV replication is limited by the rate of generation of new permissive macrophages. The steady-state viremia attained about 5 weeks after infection reflects a balance between LDV replication in permissive macrophages as they arise and LDV inactivation and clearance.     

Immunoglobulins that bind to uncoated ELISA plate surfaces: appearance in mice during infection with lactate-dehydrogenase-elevating virus and in human anti-nuclear antibody-positive sera

Immunoglobulins present in the blood plasma of mice infected with lactate-dehydrogenase-elevating virus (LDV) were found to bind strongly in the presence of 0.05% Tween 20 to the uncoated surfaces of wells of certain ELISA plates with previously recognized high protein-binding capacity. The binding was readily distinguishable from non-specific background binding of immunoglobulins present in normal mouse plasma. The binding components absorbed to protein A and had molecular weights in the 150-300 kDa range. Binding of the purified IgG fraction was progressively inhibited by increasing the concentration of Tween 20 in the diluent and by preincubation of the fraction at pH 3-4 for 10 min. The appearance of plate-binding IgM and IgG during LDV infection corresponded approximately with previously reported time courses of appearance of IgM- and IgG-containing circulating immune complexes and of specific IgM and IgG anti-LDV antibodies in LDV-infected mice. We conclude that complexes of IgG and IgM with LDV antigens have a much higher affinity for ELISA plates with high protein-binding capacity than uncomplexed immunoglobulins. Immune complexes did not significantly bind to ELISA plates with low protein-binding capacity, which, therefore, are suitable for measuring specific antiviral antibodies. Preliminary experiments with human anti-nuclear antibody-positive serum samples demonstrated markedly elevated non-specific binding of immunoglobulins to high-binding-capacity ELISA plates.     

Immune complexes that bind to ELISA plates not coated with antigen in mice infected with lactate dehydrogenase-elevating virus: relationship to IgG2a- and IgG2b-specific polyclonal activation of B cells.

We have further investigated the nature of IgG-containing complexes of 150-300 kD that rapidly appear in the circulation of mice of various strains after infection with lactate dehydrogenase-elevating virus (LDV) and are recognized and quantitated by their binding in the presence of 0.05% Tween 20 to certain enzyme-linked immunosorbent assay (ELISA) plates with high protein affinity that have not been coated with protein antigen (5). These binding complexes have been found to contain primarily IgG2a or, in some mice, IgG2b. Their isotype specificity and time course of formation correlated with those of the polyclonal production of immunoglobulins in these mice, as measured by increases in total IgG2a or IgG2b in the circulation. In contrast, anti-LDV antibodies exhibited much broader isotype specificities in all mouse strains investigated. Depletion of BALB/c mice of CD4+T cells or lack of T cells in nude Swiss mice only partly reduced the polyclonal activation of B cells and the formation of ELISA plate-binding complexes, whereas anti-LDV antibody formation was completely blocked. Only a small proportion of the total IgG2a or IgG2b formed as a result of the LDV-induced polyclonal activation of B cells was recovered in plate-binding complexes, which sedimented in sucrose density gradients between 150 and 300 kD. Diverse monoclonal antibodies of different IgG isotopes did not bind to the plates at concentrations at which LDV-induced immune complexes exhibited binding activity. We suggest that the LDV-induced immune complexes do not contain anti-LDV antibodies, but are complexes of auto-antibodies and self-antigen(s). However, additional features must be responsible for the high affinity of these complexes for ELISA plates since various immune complexes formed in vitro failed to bind to the plates, and binding activity of the immune complexes formed in LDV-infected mice could not be regenerated in vitro once the complexes had been dissociated by a low pH treatment.     

Polyclonal B cell activation of IgG2a and IgG2b production by infection of mice with lactate dehydrogenase-elevating virus is partly dependent on CD4+ lymphocytes

Concentrations of IgM and IgG isotypes were determined by capture ELISA in plasma of Swiss, BALB/c and C58/M mice. Plasma IgG isotype concentrations, especially of IgM, IgG1 and IgG2a, varied considerably between mouse strains, batches of mice of the same strain and individual mice and as a function of age. Infection of the mice with LDV, which is known to replicate primarily in a subpopulation of macrophages, consistently resulted in a rapid elevation of plasma IgG2a (or of IgG2b in some Swiss nu/+ mice), but no plasma IgG increases were observed in mice immunized with inactivated LDV. Plasma IgG2a elevation after LDV infection was greatly delayed and reduced by depletion of the mice of CD4+, but not of CD8+, T cells by administration of protein-G-purified anti-CD4 or anti-CD8 mAbs, and completely inhibited by repeated treatment of the mice with cyclophosphamide. Treatment with anti-CD4 mAbs, or cyclophosphamide also greatly reduced the production of anti-LDV antibodies, while not significantly affecting the replication of LDV in these mice. Nude Swiss mice also failed to produce anti-LDV antibodies, though supporting normal LDV replication. Plasma IgM, IgG1, IgG2a and IgG2b levels increased in LDV-infected nu/nu mice, but similar changes were observed in uninfected mice. The results indicate that the LDV-induced polyclonal activation of B cells requires productive LDV infection of mice and is, at least partly, dependent on functioning CD4+ cells. They suggest that productive infection of the LDV-permissive subpopulation of macrophages leads to the activation of CD4+ T lymphocytes of subset 1 and their Spleen cells from 5-day LDV-infected BALB/c mice incorporated [3H]thymidine 2-3 times more rapidly in vitro than spleen cells from companion uninfected mice, whereas their responses to concanavalin A and lipopolysaccharide were reduced 60-70%.     

Immune response to lactate dehydrogenase-elevating virus: serologically specific rabbit neutralizing antibody to the virus.

A rabbit was immunized with large amounts of the lactate dehydrogenase-elevating virus (LDV) over a 9-month period. The plasma from this rabbit possessed an anti-LDV IgG titer of 1:80,000 as measured by the enzyme-linked immunosorbent assay (ELISA) method and a neutralizing titer of 1:1,000 for the homologous strain of LDV. LDV neutralization at 4 degrees C followed single-hit kinetics. In contrast, mouse anti-LDV IgG in plasma of chronically LDV-infected mice failed to neutralize LDV at 4 degrees C and neutralization at 37 degrees C was slow, biphasic, and inefficient compared with the neutralization caused by rabbit anti-LDV IgG, even though high levels of anti-LDV IgG were detectable in mouse plasma by the ELISA method. Rabbit anti-LDV IgG neutralized one heterologous strain of LDV as rapidly as it did the homologous strain, but failed to significantly neutralize five other strains of LDV, all of which were originally isolated from different mouse strains bearing transplantable tumors. The results indicate clear serological differences between LDV strains. Cross-reactions between the strains, however, were observed by ELISA, using the antibody induced during persistent infection of mice with each LDV strain. Immunoglobulin M (IgM) from mice infected for 15 days with the various strains bound equally to our LDV strain. IgG obtained from 2-month-infected mice also cross-reacted, but to a varying extent which partly correlated with the specificity detected by neutralization. Both rabbit and mouse anti-LDV IgG enhanced the infectivity of LDV at a low multiplicity of infection for primary cultures of peritoneal mouse macrophages.     

Glucocorticoid regulation of lactate dehydrogenase-elevating virus replication in macrophages

Lactate dehydrogenase-elevating virus (LDV) is a macrophage-tropic arterivirus which generally causes a persistent viremic infection in mice. LDV replication in vivo seems to be primarily regulated by the extent and dynamics of a virus-permissive macrophage population. Previous studies have shown that glucocorticoid treatment of chronically LDV-infected mice transiently increases viremia 10-100-fold, apparently by increasing the productive infection of macrophages. We have further investigated this phenomenon by comparing the effect of dexamethasone on the in vivo and in vitro replication of two LDV quasispecies that differ in sensitivity to immune control by the host. The single neutralizing epitope of LDV-P is flanked by two N-glycans that impair its immunogenicity and render LDV-P resistant to antibody neutralization. In contrast, replication of the neuropathogenic mutant LDV-C is suppressed by antibody neutralization because its epitope lacks the two protective N-glycans. Dexamethasone treatment of mice 16 h prior to LDV-P infection, or of chronically LDV-P infected mice, stimulated viremia 10-100-fold, which correlated with an increase of LDV permissive macrophages in the peritoneum and increased LDV infected cells in the spleen, respectively. The increase in viremia occurred in the absence of changes in total anti-LDV and neutralizing antibodies. The results indicate that increased viremia was due to increased availability of LDV permissive macrophages, and that during a chronic LDV-P infection virus replication is strictly limited by the rate of regeneration of permissive macrophages. In contrast, dexamethasone treatment had no significant effect on the level of viremia in chronically LDV-C infected mice, consistent with the view that LDV-C replication is primarily restricted by antibody neutralization and not by a lack of permissive macrophages. beta-Glucan, the receptor of which is induced on macrophages by dexamethasone treatment, had no effect on the LDV permissiveness of macrophages.     

Isotypically restricted activation of B lymphocytes by lactic dehydrogenase virus

Lactic dehydrogenase elevating virus (LDV) was found to selectively stimulate IgG2a synthesis in infected mice. Within one week after infection, the production of IgG2a increased nearly 50-fold whereas that of IgM, IgA, IgG1 and IgG3 remained virtually unchanged. IgG2b synthesis was also enhanced but to a lesser extent. Several observations suggested that this stimulation of IgG2 production resulted from a polyclonal B cell activation: (a) the isoelectric focusing patterns of IgG2a before and after LDV infection were exactly the same, (b) the frequency of clones with anti-LDV activity in hybridoma collections derived from infected mice was extremely low (less than 4/1000) and (c) the proliferative response elicited by LDV in unsensitized animals was comparable with that induced by lipopolysaccharide. The effect of LDV on immunoglobulin synthesis was drastically reduced in nude mice but was not affected by the X-linked B lymphocyte defect of animals carrying the xid mutation.     

Plasmodium falciparum: natural and experimental transmission-blocking immunity

Fc-dependent regulation of humoral immune responses was investigated by immunization of BALB/c mice with immune complexes. These complexes were composed of DNP- and PC-conjugated KLH or Ficoll, and monoclonal T15 idiotype-positive anti-PC antibodies of different isotypes but indistinguishable V-region properties. Since the response to DNP was analyzed, effects due to masking of antigenic determinants by anti-PC antibodies are excluded. The responses to free and complexed antigens showed significant differences in the proportions of DNP-specific IgM, IgG1, IgG2 and IgG3 antibodies. Complexes containing the T-dependent carrier KLH elicited serum antibodies to DNP with significantly decreased IgM levels, irrespective of the isotype in the complex. Under these conditions, however, DNP-specific IgG classes were augmented to various extents, depending on the isotype in the complex. In the case of the T-independent carrier Ficoll, only complexes with IgM or IgG3 suppressed the IgM response. Moreover, immunization with complexes composed of IgM or IgG2a led to a significant decrease of DNP-specific IgG1. In contrast to changes induced by antibodies bound to the T-dependent antigen, immunization with T-independent complexes did not enhance the production of any of the immunoglobulin isotypes.     

Endotoxin-induced auto-immunity in mice. I. Time and dose dependence of production and serum levels of antibodies against bromelain-treated mouse erythrocytes and circulating immune complexes

Injection of mice with endotoxin results in the formation of auto-antibodies and the appearance of soluble immune complexes in the blood. In this study the relationship between the production and serum levels of autohaemolysins and circulating immune complex titres was investigated. Immune complexes were detected by a solid-phase C1q-binding assay and found to contain antibodies of the IgM, but not of the IgG class. Individual mice showed marked differences as to their splenic plaque-forming cells, serum autohaemolysin, and circulating immune complex responses, both in kinetic studies and dose-response experiments. The dissociation between production and serum levels of auto-antibodies was ascribed to extrasplenic synthesis or a disproportionate production per plasma cell. The independent behaviour of the circulating immune complex response could, at least partially, be attributed to differential complement-dependent clearance from the circulation. The implications of our findings for the laboratory diagnosis of auto-immunity at the blood level are being discussed.     

Detection and composition of immune complexes in experimental African trypanosomiasis.

Humoral immune responses in experimental African trypanosomiasis were assessed in rabbits infected with Trypanosoma rhodesiense. Immune complexes as measured by a Clq binding assay or a Clq solid-phase assay were detected by day 10 and increased progressively through day 28. Immune complexes analyzed by double diffusion in agar or enzyme immunoassay contained immunoglobulin M (IgM) and IgG consistently, and usually C3. In one serum, immune complexes contained trypanosomal antigens in addition to C3, IgG, and IgM. By sucrose density gradient ultracentrifugation Clq-binding immune complex-like materials were shown to contain IgG, IgM, and C3, sedimentating as intermediate (between 7S and 19S) or as higher-molecular-weight (greater than 19S) aggregates. Serum IgM and IgG antibodies to trypanosomes were measured by enzyme immunoassay. IgM antibodies were detected by day 7, rose to peak by day 14, and declined slowly thereafter. IgG antibodies were detected by day 14 and continued to rise through day 32. Total IgM and IgG measured by radial immunodiffusion paralleled the corresponding changes in antibody levels. Host immune responses, in part directed to trypanosomal antigens, produced circulating immune complexes containing bound C3; these may be deposited in tissue or may serve as a serological marker of immune complex-mediated tissue injury.     

Non-Responsiveness to Mercury-Induced Autoimmunity in Resistant DBA/2 Mice is not Due to Immunosuppression or Biased Th1-Type Response

Mercury can induce systemic autoimmunity in susceptible mouse strains characterized by a T-cell-dependent polyclonal B-cell activation, increased serum levels of IgG1 and IgE antibodies, production of autoantibodies, and the formation of immune complexes in the kidneys. However, certain resistant mouse strains do not show any of the autoimmune manifestations after mercury injection. Th1/Th2 dichotomy has been proposed to be responsible for resistance and susceptibility, respectively. Immunosuppression has also been suggested in resistant animals after mercury injection. To test whether immunosuppression or a biased Th1-type response was induced by mercury in resistant DBA/2 mice, we injected DBA/2 mice with mercury for 1 or 3 weeks and then immunized the mice with horse red blood cells (HRBCs) to study whether the subsequent humoral response to HRBCs was inhibited or skewed to the production of antibodies of IgG2a isotype switched by Th1-type cytokines. We found that there was no reduction of the number of splenic antibody-producing cells in the subsequent response to HRBCs compared with saline-treated mice. By haemagglutination tests, the titers of HRBC-specific antibodies were the same after HRBCs injection in both mercury- and saline-treated DBA/2 mice. There was no increase in total serum IgG2a antibody. Sera of both mercury- and saline-treated mice immunized with HRBCs showed high titres of specific IgM, IgG1 and IgG2a anti-HRBCs antibodies. Surprisingly, 3-week treatment with mercury induced a reduction in the titres of specific IgG2a anti-HRBCs antibodies in DBA/2 mice after immunization with HRBCs. Our results demonstrated that mercury did not induce a general immunosuppression or a biased Th1-type immune response in resistant DBA/2 mice. The nonresponsiveness in mice resistant to mercury-induced autoimmunity must be due to some other unknown mechanism(s).     

Generation of memory cells. III. Antibody class requirements for the generation of B-memory cells by antigen--antibody complexes.

Immunizing mice with preformed antigen--antibody complexes is a highly effective means of generating B-memory cells. In the present study we have compared the capacity of mouse anti-dinitrophenyl (DNP) IgM, IgG1, IgG2 and IgA antibodies to generate DNP-specific memory, when given in complex with antigen (DNP-KLH: keyhole limpet haemocyanin). Two monoclonal IgM antibodies exerted no adjuvant effect, whereas a monoclonal IgA antibody was effective, IgG2 antibodies were a more powerful adjuvant than IgG1, regardless of whether anti-DNP or anti-KLH antibodies were used. Furthermore, the capacity of DNP-KLH--antibody complexes to localize in splenic lymphoid follicles could be ranked IgG2 greater than IgG1 greater than IgA; IgM complexes did not localize in follicles. These results correlate well with data (presented elsewhere) on the capacity of these different antibodies to activate mouse complement, and confirm that C activation is an essential requirement for both follicular localization of immune complexes, and for the generation of B-memory cells. Although activation of the alternative complement pathway is sufficient to effect both processes, the results with IgG2 antibodies raise the possibility that classical pathway activation may be more effective.     

Retention of Immune Complexes by Murine Lymph Node or Spleen Follicular Dendritic Cells

Using monoclonal anti-trinitrophenyl (TNP) antibodies complexed to TNP-myoglobin-coated gold particles, we analysed at the ultrastructural level the retention by follicular dendritic cells (FDC) of immune complexes containing various antibody isotypes. Gold-labelled immune complexes were injected subcutaneously or intravenously into naive mice and, after 24 h, germinal centres of draining lymph nodes or spleen were examined by electron microscopy. FDC generally retained complexes containing IgG2a and IgG2b better than those formed with IgG1 or IgG3. IgM was rarely retained. FDC isolated from lymph nodes or spleens were incubated in vitro with gold-labelled complexes in a serum-free medium. IgG2a and IgG2b complexes were also retained in vitro in large quantities by FDC; IgG1 and IgG3 complexes were retained in smaller quantities or in highly variable quantities compared with IgG2; IgM complexes were rarely seen on FDC. There was no difference between FDC isolated from lymph nodes or from spleen with respect to the Ig isotypes required for Fc-mediated retention of immune complexes.     

Analysis of immunoglobulin isotype blood levels, splenic B-cell phenotypes, and spleen cell immunoglobulin gene expression in mice infected with lactate dehydrogenase-elevating virus

B-lymphocyte activation was studied in mice infected with lactate dehydrogenase-elevating virus (LDV). ELISA determinations of blood total immunoglobulin levels demonstrated that, at 10 days post-infection (p.i.) with LDV, only the IgG2a isotype was elevated. DNA-excess dot-blot hybridization showed that RNA specific for IgG2a and IgA immunoglobulin isotypes was increased in the spleens of mice at 10 days p.i. with LDV. Immunoglobulin surface phenotype analysis of spleen cells at 8-10 days p.i. with LDV revealed that there was no alteration in immunoglobulin isotype-bearing cell proportions, although total spleen mass and number of cells increased during LDV infection. When blood immunoglobulins from LDV-infected mice were analyzed by two-dimensional isoelectric focusing gels, followed by specific immunoblotting for immunoglobulin isotype, the presence of new IgG2a species was observed at 10 days p.i.     

Detection of the binding of IgG2a and IgG2b on the surface of macrophages from mice chronically infected with lactic dehydrogenase virus.

This study showed that sera from mice chronically infected with lactic dehydrogenase virus (LDV) contained virus-antibody complexes (IC). IgG2a and IgG2b, but not IgG1, IgG3, IgM or IgA, were demonstrated on the surface of macrophages from chronically infected mice. These results suggest that IC in the circulation may bind to Fc receptors for IgG2a and IgG2b on the surface of macrophages and lead to the modulation of macrophage function seen in chronically LDV-infected mice.     

Comparison of receptors required for entry of Leishmania major amastigotes into macrophages.

We investigated the mechanisms of entry of amastigotes of Leishmania major from two different sources into macrophages by comparing their use of the Fc receptor (FcR), complement receptor type 3 (CR3), and mannose-fucose receptor (MFR). Amastigotes were obtained from BALB/c mice and SCID mice. FcR involvement was examined by opsonizing L. major with parasite-specific immunoglobulin G (IgG). Antiparasite IgG did not alter the uptake of amastigotes from BALB/c mice since these amastigotes had antibody bound to their surface: IgG1 was the most predominant antibody, followed by IgG2b, IgM, and IgG2a. However, opsonization with antiparasite IgG enhanced the entry of amastigotes that lacked antibody on their surface, namely, amastigotes obtained from SCID mice or from macrophages infected in vitro. These results indicate that the FcR is important for amastigote entry into macrophages. Down-modulation of FcRs onto immune complexes, however, did not reduce the entry of amastigotes containing surface-bound IgG into macrophages. Monoclonal antibodies against the CR3 inhibited the entry of amastigotes from either BALB/c or SCID mice into J774A.1 macrophage-like cells. Simultaneous blocking of FcR and CR3 further increased the inhibition of phagocytosis. Treatment of macrophages with soluble mannan or down-modulating the MFR onto mannan-coated coverslips had no effect on the entry of amastigotes from BALB/c or SCID mice. Thus, the MFR does not appear to be used by amastigotes of L. major. We show that ingestion of amastigotes appears to occur primarily through the FcR and CR3; however, additional receptors may also participate in the uptake of amastigotes.