The chemiluminescent response of human monocytes to red cells sensitized with monoclonal anti-Rh(D) antibodies

Summary Luminol-enhanced chemiluminescence (CL) was used to assess the metabolic response of human monocytes to red cells sensitized with known amounts of anti-Rh(D). Monoclonal antibodies were used to facilitate a comparison between the functional activities of IgG1 and IgG3 subclasses. The detection of CL provided a simple, rapid and semi-quantitative means of measuring monocyte response to sensitized red cells (IgG-RBC). Monocyte response to IgG3-RBC was quantitatively greater, more rapid and less susceptible to inhibition by fluid phase IgG than monocyte response to IgG1-RBC. The minimum levels of sensitization required to elicit CL from monocytes were approximately 2500 IgG3 molecules per red cell, or approximately 5000 IgG1 molecules per cell.     

Heterogeneity of IgG1 monoclonal anti-Rh(D): an investigation using ADCC and macrophage binding assays

Three monoclonal IgG1 anti-Rh(D), UCH D4, ARC 7D5 and UKTS FC3, produced by Epstein-Barr virus transformed cells from Rh(D)-sensitized individuals, were compared with polyclonal single donor anti-D sera and therapeutic immunoglobulin preparations in antibody dependent cellular cytotoxicity (ADCC) and macrophage binding tests. When assayed at equal anti-D concentrations monoclonal antibodies varied considerably in their ADCC and macrophage binding activities: only UKTS FC3 showed significant activity in both assays, but these were substantially lower than those of the polyclonal anti-D sera and immunoglobulins. When examined in different combinations the monoclonal antibodies showed little synergism in mediating red cell destruction by the effector cells. Factors which might contribute to the diverse ADCC and macrophage binding activities of the monoclonal anti-Ds of the same IgG subclass are discussed.     

Production of human monoclonal IgG antibodies against Rhesus (D) antigen.

An Epstein-Barr virus (EBV)-transformed human B-cell line ( LB4r ) producing anti-Rhesus [Rho(D) antigen] antibody was fused with a non-immunoglobulin-producing mouse-human heteromyeloma ( SHM - D33 ) and selected in hypoxanthine/aminopterin/thymidine medium containing 0.5 microM ouabain. Surviving hybrids found to secrete specific anti-Rho(D) antibody were cloned by limiting dilution. Two clones (D4-B2 and E10-C1) producing high levels (12 and 20 micrograms/ml per 10(6) cells per 24 hr, respectively) of monospecific antibody (IgG3, lambda chain) were selected for expansion and further characterization. Compared to the parental cell line ( LB4r ), these hybridoma cell lines presented several advantages: antibody production was increased 10-fold, cloning efficiency was improved, and the EBV genome was not retained. Antibody production has been stable for greater than 8 months. These human monoclonal anti-Rho(D) antibodies have demonstrated utility in routine blood-group typing. They may also prove useful in the biochemical and genetic characterization of the Rh antigen system. Most important, they offer a source of Rh-immune globulin for the prevention of Rh immunization and alloimmune hemolytic disease of the newborn.     

A human antibody to Gm (26): an antigen usually present on the gamma-3 chain of IgG when Gm (15) is absent.

An antibody to an antigen on IgG was found in the serum of a healthy American Negro, whose phenotype is Gm (1, 13, 15, 17). (When tested for Gm [1, 2, 3, 5, 6, 13, 15, 16, 17, 21].) Subsequent tests of serum samples from US blacks and whites, from Japanese, Ainu, San (Bushmen), Negros, Asiatic Indians and Jews from Cochin India demonstrated that the antibody detects an antigen that is usually present in a haplotype when Gm (15) is absent from it. This antigen had been identified using an antibody produced in a baboon. Tests of isolated myeloma proteins and of Fc and Fab fragments of IgG confirmed that the antigen [Gm (26) or Gm (u) - originally Gm (Pa)] is carried by the Fc portion of the gamma3-chain.     

Binding characteristics of anti-Rh0(D) antibodies to Rh0(D)-positive and Du red cells

The relation of human erythrocyte Rh0(D) to Du sites is an important unresolved question in the field of immunohematology. To compare the immunological reactivity of Rh0(D)-positive and Du erythrocytes, the binding characteristics of two anti-Rh0(D) antisera to human Rh0(D)- positive and Du ("low-grade") erythrocytes were studied. 14C-Protein A and direct antibody-labeled techniques were used to generate binding curves and to derive double-reciprocal plots. The results show that the number of antigen sites differ by a factor of 10 to 15 between the Rh0(D)-positive and Du red cells, but that the dissociation constants between anti-Rh0(D) and the Rh0(D) and Du antigens are indistinguishable when studied by the two labeling methods and two different anti-Rh0(D) antibodies. The extent of binding to 112 different Du samples showed a normal distribution and was independent of apparent phenotype. These data suggest immunologic identity of Rh0(D) and Du ("low-grade") sites and that the difference between the antigens of Rh0(D) and Du cells is quantitative only. The data are incompatible with the "missing mosaic" and gene interaction theories of mechanism.     

Genes associated with the G2m(23) immunoglobulin allotype regulate the IgG subclass responses to Haemophilus influenzae type b polysaccharide vaccine

We determined whether genes associated with the G2m(23) allotype, a genetic marker on IgG2 molecules, influence the subclass composition of the antibody response to Haemophilus influenzae type b polysaccharide vaccine. After immunizing 70 white adults, the geometric means (GMs) of the total, IgG, IgG1, and IgG2 antibody concentrations increased approximately 10-fold over preimmunization concentrations. There was no significant difference in the GMs of the total, IgG, or IgG1 antibody concentrations in postimmunization sera from subjects positive versus those negative for G2m(23). Adults positive for G2m(23) had a GM IgG2 concentration more than threefold higher than that of negative subjects (P less than .001). The antibody responses of 61 Amish adults immunized with type b polysaccharide vaccine were also analyzed. Those adults positive for G2m(23) had a higher GM serum IgG2 response to capsule than did those who were negative (P less than .01). These data indicate that genes associated with the G2m(23) locus regulate the IgG subclass composition of the antibody response to H. influenzae type b polysaccharide vaccine.     

Differential reactivity of rheumatoid synovial cells and serum rheumatoid factors to human immunoglobulin G subclasses 1 and 3 and their CH3 domains in rheumatoid arthritis

19S IgM rheumatoid factors (RF) are polyclonal autoantibodies that may play an important pathogenic role in sustaining inflammatory synovitis in rheumatoid arthritis (RA). RF in RA have reactivity for as-yet-uncharacterized antigenic determinants in IgG Fc. We hypothesized that qualitative differences might exist between some of these RF molecules, and that differences such as reactivity and affinity might characterize more pathogenic RF molecules. Previous observations in our laboratory indicate that RF produced by rheumatoid synovial cells (RSC) have greater reactivity with human IgG and IgG3 subclass, in contrast to serum RF, which has greater reactivity with rabbit IgG and human IgG1. These observations were made using a complement-dependent RF plaque-forming cell assay. The purpose of this study was to validate and extend those observations. Therefore, we examined the reactivity of RSC and serum RF with human and rabbit IgG and the reactivity and avidity of RSC-RF for IgG1 and IgG3 molecules and Fab, F(ab')2, and pFc' fragments thereof in a solid-phase enzyme immunoassay. In particular, we found: RSC-RF had at least twice as much reactivity with human IgG as with rabbit IgG; serum RF had approximately equal reactivity with human and rabbit IgG; RSC-RF had greater reactivity and avidity for IgG3 and IgG3 pFc' than for IgG1; and RSC-RF was nonreactive with Fab or F(ab')2 from either IgG1 or IgG3. These results suggest that the major antigenic determinant for RSC-RF resides in the CH3 domain of the IgG3 molecule. Precise characterization of this epitope may provide further insight into the etiology and pathogenesis of RA.     

Efficacy of rhesus antibodies (anti-Rh0 (D)) in autoimmune thrombocytopenia: Correlation with response to high dose IgG and the degree of haemolysis

Summary We have compared the efficacy of high-dose IgG with that of Rhesus antibodies (anti-Rh₀ (D)) in 5 patients with autoimmune thrombocytopenic purpura (3 adults and 2 children). Although only transient, high-dose IgG (0.4 g/kg×5 days) was effective in all patients (peak values 50–200×10⁹/l), whereas anti-Rh₀(D) (11–20 g/kg×5 days) led to comparable results in only 3 patients (165×10⁹/l, 72×10⁹/l, 33×10⁹/l). This response to anti-Rh₀ (D) was neither related to the degree of induced haemolysis (increase of LDH and decrease of haptoglobin) nor to the amount of IgG antibodies bound to red blood cells, as quantified by the 125-I-antiglobulin test. A decrease of platelet-associated IgG was recorded in 3 patients: 2 of them showed an improvement of platelet counts and in one of them there was no response.We conclude that the therapeutic response of high-dose IgG and anti-Rh₀ (D) is independent of the degree of induced haemolysis and may not be predicted from the effectiveness of either therapy alone.     

Immunological follow-up of 17 patients with rheumatoid arthritis treated in vivo with an anti-T CD4+ monoclonal antibody (B-F5)

Seventeen patients with steroid-refractory rheumatoid arthritis were treated with a monoclonal antibody: anti-T CD4/B-F5 (IgG1) for 10 days. The daily dose was 20 mg. No severe side effects were observed and clinical improvement was seen in 15 patients, accompanied by a steep decline in C reactive protein levels. This improvement persisted as long as 12 months in 3 patients. A decline in lymphocyte counts was observed 2 hours after infusion. CD3+, CD4+, CD8+ and B cells were affected. Monocyte levels also decreased, whereas NK cell levels remained unchanged. After 24 hours a subsequent recovery of lymphocyte cell numbers made it possible to return to pre-treatment levels. Residual CD4+ cells coated with CD4 antibody were sporadically found even if residual antibody could be detected in the serum. These results indicate insufficient mAb concentrations. No patients developed detectable anti-mouse Ig antibodies during the treatment period, but 5 patients developed antibodies 15 to 30 days after the end of the treatment. Proliferative responses (mainly the response to ConA) were reduced at the end of the treatment. One month later the proliferative response returned to pre-treatment levels. mAb treatment did not induce long lasting cell activation, as indicated by the low levels of CD25+ or DR+ cells. Soluble IL2 receptor levels were significantly higher before treatment, but did not change after treatment. Soluble CD8 and soluble CD4 molecules were also more numerous before treatment and this increase was correlated with clinical parameters. Of interest was the correlation between the variations in soluble CD8 and the Ritchie index during treatment. The increased levels of serum TNF alpha and IL6 were not modified by treatment. A randomized study now appears necessary to prove the efficacy of the treatment. Such a study would also provide biological data and thus help to define factors predictive of a response in this heterogeneous disease.     

The mechanism involved in the conversion of thyrotropin receptor-bound blocking-type immunoglobulin G (IgG) to the stimulating-type by anti-human IgG antibodies

It has been reported that the addition of antibody (Ab) against human immunoglobulin G (IgG) converts TSH receptor-bound blocking-type IgG to stimulating-type IgG. However, the detail of converting mechanism remains unclear. In this study we examined the mechanism involved in this conversion using FRTL-5 cells. Blocking-type IgG was obtained from a patient with hypothyroidism. FRTL-5 cells were first incubated with IgG solution, then washed with PBS and exposed to antihuman IgG Ab. The effect of antihuman IgG Ab on converting activity was dose dependent. Maximal stimulation of cAMP was achieved with an antiserum dilution of 1:75. It seems likely that antimicrosomal Ab does not interfere with cAMP production, since IgG with a high anti-hemagglutination antibody titer did not show converting activity. Of the several kinds of antibodies tested, Ab against human IgG-Fab fragment was the most effective in converting ability, while the least effective were those against human IgG-Fc fragment. Although the divalent F(ab')2 fragment of antihuman IgG was significantly more effective in its converting ability than the monovalent Fab fragment, the Fab fragment itself also converted blocking IgG to the stimulating type in a dose-dependent manner. Accordingly, receptor cross-linking or aggregation does not play a major role in promoting this converting phenomenon. When cells were first exposed to blocking-type IgG and then to both antihuman IgG Ab and bovine TSH, cAMP production was much greater than the sum of each alone. However, anti-IgG Ab alone did not affect the binding of blocking-type IgG to receptor. These results suggest that the addition of antihuman IgG Ab not only converts blocking-type IgG to the stimulating type but also recovers TSH activity via a postreceptor step. Forskolin, like TSH, showed an additive effect on cAMP stimulatory action with antihuman IgG. In contrast, cholera toxin and antihuman IgG Ab were not additive. The reason for this discrepancy remains unknown. In summary, our observation indicates that 1) the converting phenomenon is induced via IgG-TSH receptor complexes; 2) the mechanism aside from receptor aggregation, i.e. the recognition of a critical domain in TSH receptor molecule, seems necessary for promoting converting phenomenon; and 3) the addition of antihuman IgG Ab affects a postreceptor step via TSH receptor structures that differ from the TSH-binding site.     

Hypotensive effect of a human anti-renin monoclonal antibody (4G1D8) in the sodium-depleted alert marmoset

A dose-response relationship was involved after an intravenous bolus of a human antirenin monoclonal antibody (4G1D8), in sodium depleted marmosets. The sodium depletion (furosemide: 30 mg/kg/d for 2 days) was used to potentiate the contribution of the renin-angiotensin system in the blood pressure (BP) control. To record BP and inject the antibody, 2 catheters were implanted the day before the experiment. The plasma renin activity (PRA) was measured by the RIA of angiotensin I after an incubation of plasmas for 1 hour at pH 7.4. The sodium depletion induced a dramatic increase of PRA (63.68 +/- 20.03 ng/ml/h of angiotensin I compared to 2.96 +/- 1.03; p less than or equal to 0.01; n = 13). The basal BP was 102.6 +/- 2.4 mmHg (n = 17). The maximal fall in BP was noted at about 30 min for the three groups of animals treated by 4G1D8; it was -7.5 +/- 4.3 mmHg at the dose of 0.01 mg/kg (n = 4), -21.3 +/- 3.8 mmHg (p less than or equal to 0.01) at 0.10 mg/kg (n = 4), and -27.5 +/- 1.4 mmHg (p = 0.10) at 0.24 mg/kg (n = 4). At the 0.10 and 0.24 mg/kg doses, the hypotension was lasting (greater than 3 h). PRA was strongly inhibited and HR was little modified. A dose-response relationship with a human antirenin monoclonal antibody, 4G1D8, provides a very interesting pharmacological model for a comparative study of renin inhibitors.     

Immunoglobulin G (IgG) responses to Plasmodium falciparum glycosylphosphatidylinositols are short-lived and predominantly of the IgG3 subclass

The induction of neutralizing immunity to Plasmodium falciparum toxins by vaccination has been proposed as a preventive strategy to limit the severity of malaria. For this approach to be successful, generation of a sustained immune response would be necessary. This study shows that immunoglobulin G (IgG)-subclass responses elicited by the proposed P. falciparum toxin glycosylphosphatidylinositol (GPI) in Papua New Guinean subjects 5-60 years old predominantly involve IgG(3), with a lesser contribution from IgG(1) and an absence of IgG(2) and IgG(4). IgG(3) levels declined sharply within 6 weeks of pharmacological clearance of parasitemia in all subjects, whereas a significant decrease in IgG(1) levels was seen only in subjects < or =19 years old. Because the natural antibody response to P. falciparum GPIs is skewed toward the short-lived IgG(3) subclass, a vaccination strategy with GPI analogues would likely require augmentation by costimulatory molecules, to induce a more persistent anti-GPI response.     

Characterization of a monoclonal IgMK (IgMGAS) anti-Gd cold agglutinin (CA). Its coexistence with a monoclonal IgG3K (IgGGAS) without CA activity that might be clonally related to IgMGAS

A monoclonal IgMK (IgMGAS) cold agglutinin (CA) of the infrequent anti- Gd specificity, found in a patient with Waldenstrom macroglobulinemia, has been characterized. IgMGAS uses a VH gene homologous (93.7%) to the reported VH251 germ-line, one of the two functional genes of the VH5 family, with differences in both framework regions and complementary determining regions (CDR). The VL gene is homologous to the reported 15AVK1 germ-line gene, recently described in an anti-i CA, with differences mostly clustered in CDR. In the patient's serum, IgMGAS coexisted with a monoclonal IgG3K (IgGGAS) that lacked CA activity but expressed the private idiotopes found on IgMGAS. Both Ig lacked reactivity with antibodies detecting VH1 or VHIII or VKIII subgroup regions or the VH4-21 gene product that is expressed by anti-I/i CAs. The K chains from both Igs showed the same isoelectrical mobility. Moreover, the k chains from both serum Igs showed the same N-terminal amino acid sequence. This sequence was identical to that predicted by the nucleotide sequence of VK1GAS gene segment, including one discrepancy at position 15 (Ile for Val) with respect to the consensus VK1 subgroup regions. Although these data do not exclude a possible independent clonal origin, they are consistent with the notion that IgGGAS might be clonally related to IgMGAS.     

IgG and IgA antibody titers against human heat-shock protein (hsp60) in sera of rheumatoid arthritis and osteoarthritis patients

 To learn whether heat-shock proteins (HSP) are involved in the pathogenesis of rheumatoid arthritis (RA), antirecombinant human heat-shock protein 60 (hsp60) IgG and IgA in sera of RA and osteoarthritis (OA) patients were investigated. Only the anti-hsp60 IgG titer of seropositive (RF-positive) patients was found to be elevated. Although RF titers of the sera of seropositive RA patients were increased, there was no correlation between the individual anti-hsp60 IgG titer and the corresponding RF titer. In contrast, all the anti-hsp60 IgA titers of the sera of OA, seronegative RA, and seropositive RA patients were found to be elevated. Among them, only the serum IgA concentration of seropositive RA patients was increased. Thus, it was suggested that the increased anti-hsp60 IgG reflects the pathogenesis of RA and its activity. It was also suggested that the increased anti-hsp60 IgA response reflects an involvement of hsp60 in the pathogenesis of arthritides rather than the pathogenesis of RA. Received: August 8, 2001 / Accepted: June 5, 2002 Acknowledgments This study was supported in part by a grant-in-aid from the Japanese Ministry of Education, Science, and Culture. We thank Dr. Yashima for kindly providing normal adult serum samples. We thank Drs. Nishimiya and Hiraoka for technical help. Correspondence to:S. Watanabe     

IgG and IgA antibody titers against human heat-shock protein (hsp60) in sera of rheumatoid arthritis and osteoarthritis patients

Abstract

To learn whether heat-shock proteins (HSP) are involved in the pathogenesis of rheumatoid arthritis (RA), antirecombinant human heat-shock protein 60 (hsp60) IgG and IgA in sera of RA and osteoarthritis (OA) patients were investigated. Only the anti-hsp60 IgG titer of seropositive (RF-positive) patients was found to be elevated. Although RF titers of the sera of seropositive RA patients were increased, there was no correlation between the individual anti-hsp60 IgG titer and the corresponding RF titer. In contrast, all the anti-hsp60 IgA titers of the sera of OA, seronegative RA, and seropositive RA patients were found to be elevated. Among them, only the serum IgA concentration of seropositive RA patients was increased. Thus, it was suggested that the increased anti-hsp60 IgG reflects the pathogenesis of RA and its activity. It was also suggested that the increased anti-hsp60 IgA response reflects an involvement of hsp60 in the pathogenesis of arthritides rather than the pathogenesis of RA.     

Antiphospholipid antibodies: a disease marker in 25 patients with antinuclear antibody negative systemic lupus erythematosus (SLE). Comparison with a group of 91 patients with antinuclear antibody positive SLE

Twenty-five antinuclear antibody (ANA) negative patients with systemic lupus erythematosus (SLE) or lupus-like disease were compared to 91 ANA positive patients with SLE for clinical and biological symptoms. Cutaneous symptoms were infrequent in ANA negative patients (p less than 0.03). Thrombocytopenia (p less than 0.001), venous or arterial thrombosis (p less than 0.02) as well as cerebral infarction (p less than 0.001) were more frequent. Three types of antiphospholipid antibodies were determined by different methods; the VDRL, the lupus anticoagulant and an ELISA for IgG anticardiolipin antibody (aCL). The frequency of a positive VDRL test was significantly higher in the ANA negative group (p less than 0.05). Correlation studies suggest that the 3 methods are not redundant and detect overlapping but not identical antibodies. Of the 3 antiphospholipid antibody assays, only the IgG aCL test was significantly associated with thrombosis in the ANA negative group (p less than 0.02).     

Anti-Rho(D) IgG binds to band 3 glycoprotein of the human erythrocyte membrane

Alkali-extracted erythrocyte ghost membranes from Rho(D)-positive and Rho(D)-negative donors were incubated with human immune anti-Rho(D) IgG and nonimmune IgG. After sensitization with IgG, the integral membrane proteins were solubilized in Brij 36T nonionic detergent and chromatographed by gel filtration. There was a distinct resolution of IgG into free and membrane-complexed forms. The IgG-complexed membrane proteins were isolated by the use of a staphylococcal protein A affinity support. The protein A-bound complexes were examined for polypeptide composition by gel electrophoresis after elution. Only Rho(D)-positive membrane proteins incubated with immune anti-Rho(D) IgG revealed intact band 3. Control Rh-negative membrane proteins that had reacted with immune anti-Rho(D) IgG and the Rh-positive membranes that had reacted with nonimmune IgG showed only low molecular weight fragments of band 3 that bound nonspecifically to IgG. Arguments are presented supporting a band 3 localization for the Rh antigen.