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Keywords:

  • Alloantibodies;
  • cardiac transpalnt;
  • complement;
  • cytokines;
  • IgKO;
  • AlloAbs;
  • alloantibodies; IgKO;
  • Ig knock-out; WT;
  • wild type; mAbs;
  • monoclonal antibodies

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Alloantibodies (AlloAbs) are a clinically significant component of the immune response to organ transplants. In our experimental model, B10.A (H-2a) cardiac transplants survived significantly longer in C57BL/6 (H-2b) immunoglobulin knock-out (IgKO) recipients than in their wild-type (WT) counterparts. Passive transfer of a single 50–200-μg dose of complement-activating IgG2b AlloAbs to IgKO recipients reconstituted acute rejection of cardiac allografts. Although passive transfer of a subthreshold dose of 25 μg of IgG2b or a single 100–200-μg dose of non-complement-activating IgG1 AlloAbs did not restore acute rejection to IgKO recipients, a combination of these AlloAbs did cause acute graft rejection. Histologically, rejection was accompanied by augmented release of von Willebrand factor from endothelial cells. IgG1 AlloAbs did not activate complement on their own and did not augment complement activation by IgG2b AlloAbs. However, IgG1 AlloAbs stimulated cultured mouse endothelial cells to produce monocyte chemotactic protein 1 (MCP-1) and neutrophil chemoattractant growth-related oncogene α (KC). TNF-α augmented IgG1 induced secretion of MCP-1 and KC. These findings indicate that non-complement-activating AlloAbs can augment injury to allografts by complement-activating AlloAbs. Non-complement-activating AlloAbs stimulate endothelial cells to produce chemokines and this effect is augmented in the milieu of proinflammatory cytokines.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Organ allograft rejection is usually attributed to an acute cellular immune response characterized by parenchymal infiltration of T lymphocytes and macrophages (1). For this reason, the majority of experimental models of transplantation have concentrated on examining cellular immune responses to allografts. Nevertheless, numerous clinical studies indicate that in some patients AlloAbs contribute to acute and chronic allograft rejection. These studies have correlated the presence of circulating antibodies or complement deposition with poor graft survival (2–8). Although some investigators have attributed a causal relationship to these clinical correlations, the pathogenic mechanisms by which antibodies cause graft rejection have not been fully established. Vascular changes in both capillaries and arteries have been noted in rejections attributed to AlloAbs. Most frequently these changes include margination of neutrophils or monocytes in capillaries or fibrinoid necrosis of arteries (7,9,10).

The intensity of antibody responses to allogeneic MHC antigens varies widely depending on the donor-recipient MHC disparity and the responsiveness of the recipient. In rats, the magnitude of alloantibody responses to allogeneic blood transfusions and subsequent renal allografts was found to be genetically linked to the MHC of the recipient (11). Similarly, the antibody response to transplants varies among different recipient strains of mice and rats (12–14). In strains of rats that produce strong antibody responses to cardiac transplants, the transfer of serum containing high titers of antibodies to the transplant can break through immunosuppression and induce rejection (12,13).

The most cogent in vivo experiments have taken advantage of animals that are genetically deficient in B cells or complement components. One type of B-cell-deficient mouse has been created by targeted disruption of the membrane exon of the immunoglobulin μ chain gene (15). These immunoglobulin ‘knock-out’ (IgKO) mice have impaired B-cell development and are unable to produce antibodies. The effects of the B-cell deficiency on allograft rejection are dependent on the strain combination studied. For example, in spite of the absence of antibodies, C57BL/6 IgKO recipients acutely reject hearts transplanted from fully MHC and non-MHC disparate C3H/He donors (16). However, when a different mouse strain is selected as the donor, antibodies can overcome inadequate immunosuppression. A subtherapeutic dose of cyclosporine A prevents acute rejection of fully MHC and non-MHC disparate Balb/c cardiac allografts in C57BL/6 IgKO recipients, but not in their normal counterparts (17). Finally, in some strain combinations, T-cell immunity is not fully capable of causing acute rejection in the absence of antibodies. Approximately 85% of the cardiac allografts from B10.A, B10.BR, or B10.D2 donors survive more than 14 days in nonimmunosuppressed C57BL/6 IgKO recipients, but not in their normal counterparts (18). This deficiency can be reversed by administering appropriate AlloAbs. We documented that acute rejection of cardiac allografts was restored to IgKO recipients by passive transfer of proinflammatory IgG2b monoclonal antibodies (mAbs) against donor MHC in a dose-dependent fashion (18–20). In contrast, IgG1 AlloAbs that do not activate complement did not restore acute rejection on their own (18). The mechanisms underlying these effects of antibodies on transplants remain to be dissected.

Complement is one mediator of antibody-initiated injury that has been demonstrated to promote inflammation by using cultured vascular endothelial cells and purified complement components (21–27). These in vitro studies have demonstrated that antibody and complement have the potential to initiate a dynamic activation of endothelial cells. The immediate effect of endothelial cell activation is retraction of the plasma membrane from the underlying substrate (23), and release of preformed von Willebrand factor and P-selectin from cytoplasmic Weibel-Palade bodies to the cell surface (26). Subsequently, activated endothelial cells produce IL-1-α, IL-8 and monocyte chemotactic protein 1 (MCP-1) to recruit neutrophils and monocytes to the site of injury (28,29). This mechanism is consistent with the findings in clinical biopsies from transplants diagnosed with antibody-mediated rejection. These biopsies contain marginated platelets and neutrophils or monocytes (9,10,30–33).

The effects of antibodies that do not activate complement are less obvious and more intriguing. These antibodies can activate endothelial cells directly by cross-linking MHC molecules. Activation of endothelial cells by this mechanism leads to proliferation, increased phosphorylation of thyrosine proteins (34,35), production of growth factors or their receptors (TGF-β, PDGF and FGF), MCP-1, cytokines and adhesion molecules (34,36–40).

AlloAbs can also stimulate NK cells, neutrophils and macrophages through their Fc receptors. These cells are known to be important effectors of antibody-dependent cell-mediated cytotoxicity (ADCC) (41–43) as well as other functions triggered through Fc receptors (44). Moreover, activated macrophages can produce TNF-α and IL-1-α, which in turn can augment endothelial cell activation by complement.

The aim of the present studies was to investigate the effects of different subclasses of monoclonal antibodies against donor MHC class I antigens on allograft rejection. Furthermore, we tested the hypothesis that the passive transfer of combinations of different alloantibody subclasses may be different from the effect of these subclasses transferred separately. In this report we provide evidence that: 1) Passive transfer of a single dose of complement activating IgG2b (15–1-5P) monoclonal antibody to IgKO recipients reconstitutes acute rejection of cardiac allografts in a dose-dependent fashion; 2) although passive transfer of a non-complement activating IgG1 alone does not reconstitute acute graft rejection, it does when combined with a subthreshold dose of IgG2b AlloAbs; 3) IgG1 AlloAbs augment endothelial injury caused by IgG2b AlloAbs in vivo; and 4) IgG1 stimulates MCP-1 mRNA expression as well as secretion of MCP-1 and KC protein by mouse endothelial cells in the absence of complement.

These findings indicate that non-complement-activating AlloAbs can augment injury by complement-activating AlloAbs. Mechanistically, non-complement-activating antibodies may augment rejection by stimulating the production of chemokines from endothelial cells.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Mice

Male C57BL/6 (H-2b), B10.A (H-2a), and C57B-Igh-6tm1Cgn inbred mice were purchased from Jackson Laboratories (Bar Harbor, ME), and used at 8–12 weeks of age. C57B-Igh-6tm1Cgn mice have a targeted gene deletion in the μ chain (IgKO). This genetic deletion has been backcrossed onto C57BL/6 for 12 generations. By the 12th generation, the backcrossed mice are genetically identical to the parental strain (45). This is a critical consideration in designing properly controlled studies in transplant models, which can be greatly affected by ‘minor’ histocompatibilities that are encoded on widely distributed genes (46).

Heterotopic heart transplantation

B10.A (H-2a) hearts were transplanted into wild-type (WT) or IgKO C57BL/6 (H-2b) recipients.

Heterotopic cardiac transplantation was performed under methoxyfluorane (Pitman-Moore, Inc., Mundelein, IL) inhalational anesthesia using the standard technique as previously described (47). Briefly, the donor aorta and pulmonary artery were anastomozed to the recipient abdominal aorta and inferior vena cava, respectively. Graft function was monitored by abdominal palpation daily until rejection, which was defined as total cessation of contractions and was confirmed by direct visualization and histologic examination of the allograft.

Measurement of complement activation by C3b and C3d deposition

Activation of complement by different IgG subclasses of antibodies was measured by flow cytometry.

In this assay 50-μL aliquots containing 1.5 × 105 B10.A lymph node lymphocytes were incubated with 50 μL of mAbs specific for H2a of IgG2b and IgG1 subclasses or their isotype-matched controls for 1 h at 4 °C. Each antibody was tested at 25, 50 or 100 μg. After the cells were washed in PBA (PBS + 0.5% BSA + 0.02% sodium azide) and then in Veronal buffer (with Ca2+ and Mg2+ (Sigma, St. Louis, MO), they were suspended in Veronal buffer and incubated in the presence of 15% normal fresh mouse sera for 1 h at 37 °C on shaker. After two washes in Veronal buffer, the cells were suspended in PBA and stained for 45 min at 4 °C with rabbit antihuman C3b (Nordic Immunological Laboratories, Tilburk, the Netherlands) or rabbit antihuman C3d-FITC (DAKO, Carpinteria, CA). Both rabbit antibodies to human C3b and C3d cross-react with mouse C3b and C3d, respectively. After two washes in PBA, cells that reacted with rabbit anti-human C3b were stained with donkey anti-rabbit-FITC with no cross-reactivity to mouse proteins (Jackson Immunoresearch Laboratories, West Grove, PA) for 30 min at 4 °C. After two washes in PBA the cells were fixed in 1% formaldehyde in PBS, and analyzed using FACScan (Becton Dickinson, Mountain View, CA).

Endothelial cell line

The transformed mouse endothelial cell line SVEC4-10 was derived from C3H/HeJ mouse lymph nodes and expressed H2k MHC specificity (48). This cell line was kindly provided by Dr Michael Edidin, Johns Hopkins University, Baltimore, MD. Unless other sources are indicated, all culture media, FBS and culture supplements were purchased from Invitrogen, Carlsbad, CA. The cells were grown to confluence in tissue culture flasks in endothelial cell media DMEM supplemented with 10% FBS, 100 U/mL of penicillin, 100 μg/mL of streptomycin, 2%l-glutamine (pen/str/glu). Cells were removed from the flask surface using 0.5% Trypsin/EDTA. Between 24 and 48 h before the cells were seeded, the culture media was replaced with DMEM supplemented with 5% FBS, pen/str/glu and 10 ng/mL Basic Fibroblast Growth Factor (R & D Systems, Minneapolis, MN). Before seeding, the cells were suspended in fresh Media 199, containing 5% FBS, pen/str/glu. 1.5 × 105 cells were seeded in each well of a sterile 96-well flat bottom tissue culture plate. The total volume in each well was 200 μL. After 24 h of incubation, which was necessary to reach confluency, the media was replaced with fresh Media 199 containing 5% FBS, pen/str/glu. As indicated in other experiments, cells were cultured with 50–200 μg/mL of anti-MHC mAbs, 200 and 2000 U/mL of recombinant mouse TNF-α (specific activity 0.35–1.8 × 109 Units/mg) (BD Biosciences Pharmingen, San Diego, CA). The final volume in all test cultures was 150 μL. Plates were incubated for an additional 24 or 48 h. At the time of harvesting, culture supernatants were collected, pooled, aliquoted and stored at –30 °C for later cytokine analysis by ELISA. Cells remaining on the surface were lyzed in 150 μL of TRIZOL (Invitrogen, Carlsbad, CA) and processed for RNA isolation and cytokine analysis by real-time PCR.

Preparation of RNA

Total cellular RNA was isolated from snap-frozen lyzed cells using TRIZOL reagent according to the manufacturer's guidelines and the method described in our previous studies (18).

Real-time PCR

Total RNA was prepared from endothelial cell cultures and then reverse-transcribed into cDNA as described in our previous studies (18).

The cDNA was then analyzed for cytokine mRNA expression by real-time PCR methods using Pre-Developed TaqMan Assay Reagents containing specific target primers and probes for TNF-α, IL-1-α and MCP-1 (PE Applied Biosystems, Foster City, CA). Data were collected with Sequence Detector software (ABI 7700 thermocycler, PE Applied Biosystems) from which an amplification plot was generated. From this plot the threshold value (CT) was calculated, representing the PCR cycle value at which fluorescence was detectable above an arbitrary threshold. Relative gene expression within each group was calculated using the CT method. The amount of target gene was normalized to the endogenous control housekeeping gene GAPDH and relative to calibrator. All experiments were repeated in triplicate.

Histology and immunohistology

Full cross sections through the center of cardiac grafts obtained at the time of sacrifice were frozen in OCT. Adjacent cross-sections were fixed in acidic 60% methanol +10% glacial acetic acid +30% water or in 10% formalin, embedded in paraffin, and sectioned at 7 microns. Rejection was assessed on sections that were stained with hematoxylin and eosin. Von Willebrand factor expression was localized by immunoperoxidase staining with an affinity purified polyclonal rabbit antibody to human von Willebrand factor (DAKO), which cross-reacts with its mouse homolog.

Passive transfer of monoclonal antibodies

All mouse mAbs, except clone F8-12–13, were obtained from ATCC. The following mAbs that react with B10.A (H-2a) were used: IgG2b clone 15–1-5P (anti-H-2KkDk), and IgG1 clone AF3-12.1.3 (anti-H2-Kk). Isotype controls IgG2b (clone MPC 11 OUA) of unknown antigenic reactivity, and IgG1 (clone F8-11–13) (Serotec, Oxford, UK) were used. Purified mAbs were prepared from culture supernatants using protein-G columns. Immunoglobulin knock-out cardiac allograft recipients were injected intravenously with IgG2b (clone 15–1-5P) at doses of 200, 100, 50 and 25 μg. IgG1 (clone AF3-12.1.3) was injected at doses of 200 μg and 100 μg. Control cardiac allograft recipients were injected with the same dose of isotype-matched control mAbs. Injections were performed i.v., 3 or 10 days after transplantation.

Measurement of MCP-1 and KC levels in EC culture supernatants

Monocyte chemotactic protein 1 (MCP-1) and KC levels in EC culture supernatants were assessed by ELISA using plates precoated with specific antibodies to MCP-1 and KC (R & D Systems, Minneapolis, MN). Both assays were conducted according to the manufacturer's recommended protocols. The intensity of the enzymatic reaction was measured at 450 nm in an E Max Microplate Reader 2100R (Molecular Devices Corporation, Sunnyvale, CA).

Statistical analysis

Survival time data in WT and IgKO cardiac allograft recipients was analyzed using a log-rank test in Kaplan-Meier analysis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

B10.A cardiac allograft survival in C57BL/6 IgKO recipients

In our previous studies we documented that cardiac allografts from B10.A (H-2a) survived longer in C57BL/6 IgKO than in their WT counterparts (18). B10.A donors differ from C57BL/6 recipients at the entire H-2 and at one minor histocompatibility locus, H-9. B10.A cardiac allografts were rejected within 7–14 days (n = 18) in WT C57BL/6 recipients. In contrast, 85% of the cardiac allografts from B10.A donors survived more than 14 days in C57BL/6 IgKO recipients (n = 39). This difference in survival was significant (p < 0.0001) by log-rank test in Kaplan-Meier plot analysis.

Effects of passive transfer of complement-activating IgG2b or non-complement-activating IgG1 mAbs specific for donor MHC antigens on cardiac allograft rejection in IgKO mice:

Passive transfer of 100 μg of complement-dependent IgG2b mAbs (clone 15–1-5P) 3 days after transplantation did not cause IgKO recipients to reject their hearts within 2 days (n = 8) (Table 1). Only modest histological differences were found in cardiac allograft recipients 24 h after injection with mAbs specific for donor MHC (n = 2) and isotype-matched mAbs (n = 2). Immunohistology performed 24 h after mAb injection demonstrated that von Willebrand factor was released from the endothelial cells in four of eight large arteries in the IgG2b-treated recipients, whereas abundant von Willebrand factor was retained in all of the arterial endothelium of the control IgKO recipients. There was focal platelet adherence in large arteries and focal platelet plugging of capillaries. All of these grafts had only mild to moderate interstitial cellular infiltration. Thus, 100 μg of complement-dependent IgG2b mAb is insufficient to cause rejection of a fresh allograft. This is in contrast with the results when mAbs were injected 10 days after transplantation. This time corresponds with ongoing cellular and humoral responses in WT controls, as shown in our previous studies (18).

Table 1. Passive transfer of allospecific monoclonal antibodies into C57BL/6 immunoglobulin knock-outrecipients with well-functioning B10.A cardiac allografts 3 and 10 days after transplantation
Dose (μg)Allo mAb# Rej. within 2 days1Control mAb# Rej. within 2 days1
  1. 1Rejection scored as acute graft failure within 2 days after monoclonal antibody treatment.

  2. 2Number of grafts rejected between 10 and 12 days after transplantation by untreated control recipients.

3 days after transplantation
100IgG2b (15–1-5P)0/8IgG2b (MPC11OUA)0/2
10 days after transplantation
ControlsNone2/352 
25IgG2b (15–1-5P)0/4IgG2b (MPC11OUA)0/2
50–200IgG2b (15–1-5P)7/7IgG2b (MPC11OUA)0/4
100–200IgG1 (AF3-12.1.3)0/5IgG1 (F8-11–13)0/3
25+100IgG2b (15–1-5P)12/12IgG2b (MPC11OUA)0/6
 +IgG1 (AF3-12.1.3) +IgG1 (F8-11–13) 

A dose of 50–200 μg of IgG2b had no immediate effect on graft function at 10 days, but caused irreversible rejection within 2 days (n = 11) (Table 1). As a result, this treatment protocol restored acute rejection (11–12 days after transplantation) of B10.A cardiac allografts in C57BL/6 IgKO recipients. This effect was dose-dependent. Injection of a low dose of 25 μg of IgG2b (n = 4) did not cause irreversible rejection for up to 4–6 days after injection (Table 1). However, the function of cardiac allografts declined over the 4-day observation period. Recipients injected with 25–200 μg of isotype-matched control antibodies maintained allografts with undiminished function (n = 12).

Immunohistology confirmed that IgG and C3 were deposited in the rejected cardiac allografts after passive transfer of IgG2b. The passive transfer of these mAbs also caused extensive aggregates of platelets that stained intensively for von Willebrand factor. These platelet aggregates occluded the arteries, capillaries and veins of the rejected allografts.

In contrast, a single intravenous injection of 100–200 μg of non-complement-activating IgG1 mAbs specific for donor MHC (clone AF3-12.1.3) did not effect cardiac allograft survival (n = 5) up to 4 days after injection when mice were sacrificed with beating hearts (Table 1). Immunohistology demonstrated that passive transfer of IgG1 mAb did not cause aggregation of platelets or release of von Willebrand factor from Weibel-Palade bodies (Figures 1 and 2).

image

Figure 1. Von Willebrand factor staining in B10.A cardiac tissue removed on days 11–14 from C57BL/6 recipients: Immunoglobulin knock-out (IgKO) recipients were injected i.v. with a subthreshold 25-μg dose of IgG2b alone, 100 μg of IgG1 alone, or with the combination of both IgG1 plus IgG2b monoclonal antibodies (mAbs) specific to donor MHC 10 days after transplantation. A single injection of 25 μg of IgG2b or 100 μg of IgG1 alone did not cause release of von Willebrand factor from Weibel-Palade bodies or aggregation of platelets. In contrast, passive transfer of 100 μg of IgG1 combined with a subthreshold 25-μg dose of IgG2b caused severe vascular injury and release of von Willebrand factor from endothelial cells in arteries, capillaries and veins.

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image

Figure 2. Extent of von Willebrand staining on platelets in arteries, capillaries and veins in nontreated wild-type (WT) or immunoglobulin knock-out (IgKO) recipients of B10.A cardiac allografts injected with 100 μg of IgG1 alone, a subthreshold 25-μg dose of IgG2b alone or with a combination of both IgG1 plus IgG2b mAbs specific to donor MHC 10 days after transplantation. Control IgKO recipients were injected with the same doses of isotype matched control mAbs. The level of von Willebrand staining on platelets in arteries, capillaries and veins in the group injected with the subthreshold 25-μg dose of IgG2b combined with 100 μg IgG1 (**) was as high as in rejecting WT (*) recipients.

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Effects of passive transfer of IgG1 combined with IgG2b mAbs on cardiac allograft rejection in IgKO mice

Because C57BL/6 WT recipients produce high titers (>64) of both allospecific IgG1 and IgG2b antibodies to B10.A cardiac transplants (18), we tested the potential synergistic or inhibitory interactions of these two subclasses of alloantibody. In this experiment, we injected a combination of 100 μg IgG1 with 25 μg IgG2b 10 days after transplantation (n = 12). Even though these doses of mAbs did not cause rejection when injected individually, this combination of two antibodies caused irreversible rejection of cardiac allografts that occurred 1–2 days after injection (Table 1).

Immunohistology demonstrated that passive transfer of a high dose of IgG1 combined with a subthreshold dose of complement-activating IgG2b caused severe vascular injury and release of von Willebrand factor from endothelial cells in arteries, capillaries and veins. This pathology was similar to that observed in WT recipients (Figures 1 and 2).

Activation of complement by IgG1 and IgG2b anti-MHC mAbs

Endothelial cells can be stimulated through their C5aR or by sublytic concentrations of the membrane attack complex formed by C5b-C9. Therefore, we tested whether IgG1 modified complement activation by IgG2b. The complement activity of the allospecific IgG2b (15–1-5P) and IgG1 (AF3-12.1.3) mAbs have been assessed in previous studies of our own as well as of other groups (18,49). In these previous studies, complement activation was tested by cytotoxicity. When heterologous rabbit serum was used as a source of complement, the IgG2b clone was very cytotoxic, but the IgG1 clone was not. As expected, cytotoxicity was a much less sensitive test when mouse serum was used as the source of complement (18).

Therefore, we developed a more sensitive test to assess the potential synergistic or inhibitory effects of IgG1 on activation of mouse complement by IgG2b. To this end, we increased the sensitivity of the target cells and detection method. The target cells were donor-strain lymph node cells that had been stimulated with Concanavalin A to express high levels of H-2. Complement activation was detected by flow cytometry for deposition of C3b and C3d. C3 is the complement component that circulates in the highest concentration in serum and its split products are deposited in the highest concentration when complement is activated by antibodies (50). Moreover, C3b and C3d bind covalently to antibodies and cell surfaces.

Cells were coated with three different concentrations of IgG2b and IgG1 (100, 50 and 25 μg/mL) and then incubated with mouse sera as the source of complement. IgG2b (15–1-5P), but not IgG1 (AF3-12.1.3), caused deposition of C3b (data not shown) and C3d (Figure 3A). These results are consistent with the cytotoxic assay shown in our previous studies (18).

image

Figure 3. (A) Deposition of C3d on B10.A lymph node lymphocytes after incubation with IgG2b (15–1-5P) and IgG1 (AF3-12.1.3) anti-MHC mAbs. Activation of complement by different IgG subclasses of monoclonal antibodies (mAbs) was measured by flow cytometry. B10.A lymph node lymphocytes were incubated with 100, 50 or 25 μg/mL of mAbs of the IgG1 or IgG2b subclasses in the presence of 15% fresh mouse sera. Then cells were reacted with rabbit antibody to human C3d-FITC, which cross-reacts with mouse C3d. The intensity of staining is shown as mode channel fluorescence (see Materials and Methods). IgG2b (15–1-5P), but not IgG1 (AF3-12.1.3), caused deposition of C3d on B10.A lymph node lymphocytes. (B) Deposition of C3d on B10.A lymph node lymphocytes induced by IgG2b (15–1-5P) alone or in combination with IgG1 (AF3-12.1.3) anti-MHC mAbs. B10.A lymph node lymphocytes were incubated with 100, 50 and 25 μg/mL of IgG2b alone (medium) or in combination with 50 or 100 μg/mL of IgG1 in the presence of 15% fresh mouse sera. IgG1 did not inhibit or augment the level of C3d deposition by IgG2b.

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The potential for IgG1 to modify complement activation by IgG2b was tested by adding 50 or 100 μg/mL of IgG1 to 25, 50 or 100 μg/mL of IgG2b. IgG1 did not inhibit or augment the level of C3b (data not shown) or C3d deposition caused by IgG2b (Figure 3B).

Effects of IgG1 and IgG2b anti-MHC mAbs on endothelial cells in the absence of complement

Because IgG1 did not measurably alter complement activation by IgG2b at any part of the dose–response curve, we assessed the complement-independent effects of IgG1 on endothelial cells. Preliminary experiments indicated that IgG1 in concentrations between 25 and 200 μg/mL caused a dose-dependent stimulation of MCP-1 mRNA expression in SVEC4-10 endothelial cells. Therefore, each mAb was tested in a concentration 50 μg/mL to assess whether IgG1 stimulated or inhibited the effects of IgG2b on endothelial cells.

The level of IL-1-α and TNF-α mRNA expression by SVEC4-10 endothelial cells was very low and their expression was not stimulated significantly by IgG1 or IgG2b alone or in combination (data not shown). However, substantial expression of MCP-1 mRNA was found. IgG1 mAbs stimulated significantly higher levels of MCP-1 than IgG2b mAbs (Figure 4). When endothelial cells were reacted with a combination of IgG1 and IgG2b AlloAbs, IgG2b did not inhibit the effects of IgG1.

image

Figure 4. Effect of IgG1 and IgG2b anti-MHC monoclonal antibodies (mAbs) on monocyte chemotactic protein 1 (MCP-1) mRNA expression by SVEC4-10 endothelial cells in the absence of complement. SVEC4-10 endothelial cells were cultured in the presence of 50 μg/mL of IgG1, IgG2b or IgG2b + IgG1 mAbs alone (medium) or with the addition of 200 or 2000 U/mL of mouse recombinant TNF-α for 48 h. The level of MCP-1 mRNA expression by SVEC4-10 endothelial cells was measured by real-time PCR. The quantity of MCP-1 mRNA was normalized relative to expression of the endogenous control housekeeping gene GAPDH using the comparative delta CT method (see Materials and Methods). Each column represents MCP-1 mRNA delta CT ± SD (data are representative of three similar experiments). IgG1 alone or in combination with IgG2b stimulated the highest levels of MCP-1 mRNA. The addition of TNF-α to endothelial cells stimulated by mAbs caused dose-dependent further stimulation of MCP-1 mRNA expression by IgG1.

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TNF-α has been reported to enhance the expression of MCP-1 by endothelial cells and TNF-α is increased in rejected hearts (data not shown). Therefore, we tested whether TNF-α enhanced stimulation of MCP-1 mRNA transcripts in endothelial cells induced by mAbs. The addition of TNF-α to these cultures increased stimulation of MCP-1 mRNA expression by IgG1. The effect of TNF-α on endothelial cells was dose-dependent in that the expression of MCP-1 mRNA increased significantly as the concentration of TNF-α was increased from 200 to 2000 units/mL (Figure 4).

Monocyte chemotactic protein 1 (MCP-1) and KC protein production was quantitated in endothelial-cell culture supernatants by ELISA. These assays confirmed that IgG1 was effective in stimulating MCP-1 production and that TNF-α augmented the production of MCP-1 induced by IgG1 mAbs (Figure 5A). Similarly, IgG1 mAbs were found to stimulate a greater production of KC by ELISA in these same culture supernatants (Figure 5B).

image

Figure 5. Effect of IgG1 and IgG2b anti-MHC monoclonal antibodies (mAbs) on monocyte chemotactic protein 1 (MCP-1) and KC protein production by SVEC4-10 endothelial cells in the absence of complement. SVEC4-10 endothelial cells were cultured in the presence of 50 μg/mL of IgG1, IgG2b or IgG2b + IgG1 mAbs alone (medium) or with the addition of 200 U/mL of mouse recombinant TNF-α for 48 h. Endothelial cell-culture supernatants were examined for the expression of MCP-1 (A) and KC (B) chemokines in ELISA. Each column represents MCP-1 or KC protein production ± SD (data are representative of three similar experiments). This assay showed that IgG1 stimulated SVEC4-10 cells to produce significant levels of MCP-1 and KC proteins. The addition of TNF-α to endothelial cell cultures caused a further increase of MCP-1 and KC protein expression.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Transplant rejection is a complex process that usually involves multiple mediators of the immune system. Clinically, AlloAbs are suspected to participate in a significant portion of acute rejections (6,7). Although the diagnosis of antibody-mediated rejection is often associated with complement deposition (6,8), this is not always a prominent feature (51,52). Our experiments elucidate mechanisms by which non-complement-activating antibodies can contribute to the acute rejection process.

We investigated the effects of different subclasses of alloantibody in vivo by transferring mouse mAbs specific for donor H-2 to IgKO recipients. The doses of AlloAb tested had no immediate effects when transferred into recipients 3 days after transplantation, but they contributed to acute graft rejection when transferred 10 days after cardiac transplantation. In our previous studies we documented that this time coincides with vigorous alloantibody production and cellular infiltration of cardiac allografts in WT recipients (18). In this milieu, the antibodies could initiate complement-mediated inflammation, stimulate macrophages through their Fc receptors (FcR) and mediate antibody-dependent T-cell cytotoxicity (ADCC). IgG2b antibodies have the capacity to elicit all of these Fc-dependent responses (18,49,53,54). Not surprisingly, passive transfer of IgG2b caused a dose-dependent irreversible rejection of cardiac allografts. High doses (50–200 μg) of allospecific IgG2b mAbs caused extensive aggregates of platelets that stained intensively for von Willebrand factor (18). These platelet aggregates occluded the arteries, capillaries and veins of rejected allografts. Previous experiments in C6-deficient rats indicate that the membrane attack complex (MAC) is critical for this type of endothelial response in allografted hearts or lungs (55,56). Similarly in vitro experiments have shown that purified components of MAC cause endothelial cells to release von Willebrand factor as well as to synthesize IL-8, MCP-1, P-selectin, E-selectin, and ICAM-1 (21,24,28,29,57,58). In contrast to IgG2b AlloAbs, the passive transfer of even high doses of non-complement-binding IgG1 AlloAbs did not cause cardiac allograft rejection, and von Willebrand factor remained confined to the storage granules of the endothelial cells in the graft (18).

As C57BL/6 WT recipients produce high titers (>64) of both allospecific IgG1 and IgG2b antibodies to B10.A cardiac transplants (18), we investigated the potential synergistic or inhibitory interactions of these two subclasses of AlloAbs in vivo. Our model demonstrated clearly that IgG1 can augment the pathogenic effects of a subthreshold dose of IgG2b.

We demonstrated that the IgG2b (15.1.P) mAb that we used for our passive transfer studies activates mouse complement. This resulted in a dose-related deposition of C3b and C3d on target cells in vitro. In contrast, the IgG1 (AF3-12.1.3) mAb did not cause any measurable C3b or C3d deposition over the same dose range. These data are important because both complement-activating and non-complement-activating mouse IgG1 antibodies have been reported (59–61). These differences have been attributed to the structure of the variable regions of IgG1 (62,63). Moreover, IgG1 has been reported to augment IgG2b- and IgG2a-induced complement-mediated lysis of erythrocytes (64). The mechanism of this synergy is incompletely understood, but it may cause steric interference of C1 inhibitor or provide more binding sites for C4b and C3b. We found no evidence that IgG1 (AF3-12.1.3) augmented C3b or C3d deposition by IgG2b. Equally importantly, the presence of AF3-12.1.3 did not inhibit C3b or C3d deposition by IgG2b.

As an alternative mechanism to complement activation, antibodies can have direct effects on endothelial cells by cross-linking MHC molecules leading to proliferation, increased phosphorylation of thyrosine proteins and induction of fibroblast growth factor receptor (34,35,40). In our in vitro studies, IgG1 alone or in combination with IgG2b AlloAbs stimulated mouse SVEC4-10 endothelial cells to produce high levels of MCP-1 and KC. These chemokines would attract macrophages and neutrophils. We also found that TNF-α augmented the effects of the IgG1 AlloAbs on MCP-1 and KC gene expression and protein production by endothelial cells. TNF-α is relevant to antibody-mediated rejection on several levels. First, both the macrophages and T lymphocytes that infiltrate grafts during rejection can produce TNF-α. Second, TNF-α can enhance complement-mediated activation of endothelial cells (24). Finally, TNF-α has been shown to augment human endothelial cell activation by antibodies to MHC class I antigens in the absence of complement (34,40).

In summary, our data can be fitted into a simple model that would account for the pathogenic effects of IgG1 AlloAbs in the presence of subthreshold doses of IgG2b AlloAbs (Figure 6). IgG1 does not interfere with complement activation by IgG2b, but it does stimulate isolated endothelial cells to secrete MCP-1 and KC in vitro. These chemokines can supplement the chemotactic effects of C5a to attract macrophages to the graft. The TNF-α produced by activated macrophages can in turn augment endothelial-cell activation by antibody cross-linking of MHC antigens and complement deposition on the endothelial cells.

image

Figure 6. Proposed mechanism for the pathogenic effects of IgG1 AlloAbs in the presence of subthreshold doses of IgG2b AlloAbs. In this model IgG1 AlloAbs bind to and activate graft endothelial cells causing secretion of monocyte chemotactic protein 1 (MCP-1), which can enhance the chemotactic effects of C5a to attract macrophages to the graft. Macrophages attracted by MCP-1 to the graft are activated by antibody and complement through FcR, CR1 and CR2. Activated macrophages produce TNF-α, which in turn augments endothelial-cell activation by antibody cross-linking of MHC antigens and complement deposition on the endothelial cells.

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Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

This work was supported by NIH grants RO1-HL63948 and PO1-HL56091.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References
  • 1
    Mason DW , Morris PJ . Effector mechanisms in allograft rejection . Ann Rev Immunol 1986 ; 4 : 119145 .
  • 2
    Feucht HE , Felber E , Gokel MJ et al. Vascular deposition of complement-split products in kidney allografts with cell-mediated rejection . Clin Exp Immunol 1991 ; 86 : 464470 .
  • 3
    Burkhardt K , Weiss M , Riethmuller G et al. Capillary deposition of C4d complement fragment and early renal graft loss . Kidney Int 1993 ; 43 : 13331338 .
  • 4
    Feucht HE , Opelz G . The humoral immune response towards HLA class II determinants in renal transplantation . Kidney Int 1996 ; 50 : 14641475 .
  • 5
    Saidman SL , Williams WW , Tolkoff-Rubin N et al. Complement activation in acute humoral renal allograft rejection: diagnostic significance of C4d deposits in peritubular capillaries . J Am Soc Nephrol 1999 ; 10 : 22082214 .
  • 6
    Michaels PJ , Fishbein MC , Colvin RB . Humoral rejection of human organ transplants. Springer seminars . Immunopathol 2003 : 25 , 119140 .
  • 7
    Racusen LC , Colvin RB , Solez K et al. Antibody-mediated rejection criteria - an addition to the Banff 97 classification of renal allograft rejection . Am J Transplant 2003 ; 3 : 708714 .
  • 8
    Feucht HE . Complement C4d in graft capillaries – the missing link in the recognition of humoral alloreactivity . Am J Transplant 2003 ; 3 : 646652 .
  • 9
    Halloran PF , Wadgymar A , Ritchie S , Falk J , Solez K , Srinivasa NS . The significance of the anti-class I antibody response. I. Clinical and pathological features of anti-class I-mediated rejection . Transplantation 1990 ; 49 : 8591 .
  • 10
    Halloran PF , Schlaut J , Solez K , Srinivasa NS . The significance of the anti-class I response. II. Clinical and pathological features of renal transplants with anti-class I-like antibody . Transplantation 1992 ; 53 : 550555 .
  • 11
    Wasowska B , Baldwin WM III , Howell DN , Sanfilippo F . The association of enhancement of renal allograft survival by donor-specific blood transfusion with host MHC-linked inhibition of IgG anti-donor class I alloantibody responses . Transplantation 1993 ; 56 : 672680 .
  • 12
    Gracie JA , Bolton EM , Porteous C , Bradley JA . T cell requirements for the rejection of renal allografts bearing an isolated class I MHC disparity . J Exp Med 1990 ; 172 : 1547 .
  • 13
    Bradley JA , Mowat AM , Bolton EM . Processed MHC class I alloantigen as the stimulus for CD4+ T-cell dependent antibody-mediated graft rejection . Immunol Today 1992 ; 13 : 434438 .
  • 14
    Russell PS , Chase CM , Winn HJ , Colvin RB . Coronary atherosclerosis in transplanted mouse hearts II. Importance of humoral immunity . J Immunol 1994 ; 152 : 51355141 .
  • 15
    Kitamura D , Roes J , Kuhn R , Rajewsky KA . B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin μ chain gene . Nature 1991 ; 350 : 423426 .
  • 16
    Elwood ET , Ritchie SC , Aruffo A et al. CD40–gp39 interactions play a critical role during allograft rejection. Suppression of allograft rejection by blockade of the CD40-gp39 pathway . Transplantation 1996 ; 61 : 49 .
  • 17
    Brandle D , Joergensen J , Zenke G , Burki K , Hof RP . Contribution of donor-specific antibodies to acute allograft rejection: evidence from B cell-deficient mice . Transplantation 1998 ; 65 : 14891493 .
  • 18
    Wasowska BA , Qian Z , Cangello DL et al. Passive transfer of alloantibodies restores acute cardiac rejection in IgKO mice . Transplantation 2001 ; 71 : 727736 .
  • 19
    Wasowska BA , Qian Z , Behrens E , Cangello D , Sanfilippo F , Baldwin WM III . Inhibition of acute cardiac allograft rejection in immunoglobulin-deficient mice . Transplant Proc 1999 ; 31 : 136 .
  • 20
    Tran KV , Layton JL , Sanfilippo F et al. Alloantibodies restore cardiac allograft rejection to IgKO mice . Transplant Proc 2001 ; 33 : 317 .
  • 21
    Hattori R , Hamilton KK , McEver RP , Sims PJ . Complement proteins C5b-C9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface . J Biol Chem 1989 ; 264 : 90539060 .
  • 22
    Benzaquen LR , Nicholson-Weller A , Halperin JA . Terminal complement proteins C5b-9 release basic fibroblast growth factor and platelet-derived growth factor from endothelial cells . J Exp Med 1994 ; 179 : 985992 .
  • 23
    Saadi S , Holzknecht RA , Patte CP , Stern DM , Platt JL . Complement-mediated regulation of tissue factor activity in endothelium . J Exp Med 1995 ; 182 : 18071814 .
  • 24
    Kilgore KS , Shen JP , Miller BF , Ward PA , Warren JS . Enhancement by the complement membrane attack complex of tumor necrosis factor-alpha-induced endothelial cell expression of E-selectin and ICAM-1 . J. Immunol 1995 ; 155 : 14341441 .
  • 25
    Kilgore KS , Flory CM , Miller BF , Evans VM , Warren JS . The membrane attack complex of complement induces interleukin-8 and monocyte chemoattractant protein-1 secretion from human umbilical vein endothelial cells . Am J Pathol 1996 ; 149 : 953961 .
  • 26
    Tedesco F , Pausa M , Nardon E , Introna M , Mantovani A , Dobrina A . The cytolytically inactive terminal complement complex activates endothelial cells to express adhesion molecules and tissue factor procoagulant activity . J Exp Med 1997 ; 185 : 16191627 .
  • 27
    Rus HG , Niculescu FI , Shin M . Role of the C5b-9 complement complex in cell cycle and apoptosis . Immunol Rev 2001 ; 180 : 4955 .
  • 28
    Flory CM , Maheswari V , Tramontini NL et al. Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8 and monocyte chemoattractant protein-1 through nuclear factor-kappa B activation . Am J Pathol 1997 ; 150 : 20192031 .
  • 29
    Torzewski J , Oldroyd R , Lachmann P , Fitzsimmons C , Proudfoot D , Bowyer D . Complement-induced release of monocyte chemotactic protein-1 from human smooth muscle cells. A possible initiating event in atherosclerotic lesion formation . Arterioscler Thromb Vasc. Biol 1996 ; 16 : 673677 .
  • 30
    Lones MA , Czer LS , Trento A , Harasty D , Miller JM , Fishbein MC . Clinical-pathologic features of humoral rejection in cardiac allografts: a study in 81 consecutive patients . J Heart Lung Transplant 1995 ; 14 : 151162 .
  • 31
    Leffell MS , King KE , Burdick JF et al. Plasmapheresis and intravenous immune globulin provides effective rescue therapy for refractory humoral rejection and allows kidneys to be successfully transplanted into cross-match-positive recipients . Transplantation 2000 ; 70 : 887895 .
  • 32
    Alejos JC , Burch C , Takemoto S et al. Humoral rejection in cardiac transplantation: risk factors, hemodynamic consequences and relationship to transplant coronary artery disease . J Heart Lung Transplant 2003 ; 22 : 5869 .
  • 33
    Magil AB , Tinckam K . Monocytes and peritubular capillary C4d deposition in acute renal allograft rejection . Kidney Int 2003 ; 63 : 18881893 .
  • 34
    Bian H , Reed EF . Alloantibody-mediated class I signal transduction in endothelial cells and smooth muscle cells: enhancement by IFN-gamma and TNF-alpha . J Immunol 1999 ; 163 : 10101018 .
  • 35
    Patterson GA , Cooper JD , Mohanakumar T , Anti HLAantibody binding to hla class I molecules induces proliferation of airway epithelial cells: a potential mechanism for bronchiolitis obliterans syndrome . J Thorac Cardiovasc Surg 2000 ; 119 : 3945 .
  • 36
    Millan MT , Geczy C , Stuhlmeier KM , Goodman DJ , Ferran C , Bach FH . Human monocytes activate porcine endothelial cells, resulting in increased E-selectin, interleukin-8, monocyte chemotactic protein-1, and plasminogen activator inhibitor-type-1 expression . Transplantation 1997 ; 63 : 421429 .
  • 37
    Vos IH , Briscoe DM . Endothelial injury: cause and effect of alloimmune inflammation . Transpl Infect Dis 2002 ; 4 : 152159 .
  • 38
    Pidwell DJ , Heller MJ , Gabler D , Orosz CG . In vitro stimulation of human endothelial cells by sera from a subpopulation of high-percentage panel-reactive antibody patients . Transplantation 1995 ; 60 : 563569 .
  • 39
    Galfayan K , Galera O , Trento A et al. The clinical significance of antibodies to human vascular endothelial cells after cardiac transplantation . Transplantation 1999 ; 67 : 385391 .
  • 40
    Harris PE , Bian H , Reed EF . Induction of high affinity fibroblast growth factor receptor expression and proliferation in human endothelial cells by anti-HLA antibodies: a possible mechanism for transplant atherosclerosis . J Immunol 1997 ; 159 : 56975704 .
  • 41
    Abdullah N , Greenman J , Pimendiou A , Topping KP , Monson JR . The role of monocytes and natural killer cells in mediating antibody-dependent lysis of colorectal tumor cells . Can Immunol. Immunother 1999 ; 48 : 517524 .
  • 42
    Macdermott RP , Nash GS , Merkle NS , Weinrieb IJ , Bertovich MJ , Formeister JF . Further evidence that antibody-dependent and spontaneous cell-mediated cytotoxicity are mediated by different processes or cell types . Immunology 1980 ; 41 : 439447 .
  • 43
    Miltenburg AMM , Meijer-Paape ME , Weening JJ , Daha MR , van Es LA , van der Woude FJ . Induction of antibody-dependent cellular cytotoxicity against endothelial cells by renal transplantation . Transplantation 1989 ; 48 : 681 .
  • 44
    van de Winkel JG , Capel PJ . Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications . Immunol Today 1993 ; 14 : 215221 .
  • 45
    Altman PL , Katz DD . Inbred and Genetically Defined Strains of Laboratory Animals . Bethesda: FASEB , 1979 .
  • 46
    Sechler JM , Yip JC , Rosenberg AS . Genetic variation among 129 substrains: practical consequences . J Immunol 1997 ; 159 : 57665768 .
  • 47
    Burdick JF , Clow LW . Rejection of murine cardiac allografts. I. Relative roles of major and minor antigens . Transplantation 1986 ; 42 : 6772 .
  • 48
    O'Connell KA , Edidin M . A mouse lymphoid endothelial cell line immortalized by simian virus 40 binds lymphocytes and retains functional characteristics of normal endothelial cells . J Immunol 1990 ; 144 : 521525 .
  • 49
    Ozato K , Mayer N , Sachs DH . Hybridoma cell lines secreting monoclonal antibodies to mouse H-2 and Ia antigens . J Immunol 1980 ; 124 : 533540 .
  • 50
    Ollert MW , Kadlec JV , David K , Petrella EC , Bredehorst R , Vogel CW . Antibody-mediated complement activation on nucleated cells. A quantitative analysis of the individual reaction steps . J Immunol 1994 ; 153 : 22132221 .
  • 51
    Exner M , Regele H , Dancea S et al. Flow cytometric crossmatching in primary renal transplant recipients with a negative anti-human globulin enhanced cytotoxicity crossmatch . J Am Soc Nephrol 2001 ; 12 : 28072814 .
  • 52
    Zachary AA , Montgomery RA , Ratner LE , Samaniego-Picota M , Kopchaliiska D , Leffell MS . Specific and durable elimination of antibody to donor HLA antigens in renal transplant patients . Am J Transplant 2004 , in press .
  • 53
    Suzuki T . Signal transduction mechanisms through Fc gamma receptors on the mouse macrophage surface . FASEB J 1991 ; 5 : 187193 .
  • 54
    Ravetch JV , Clynes RA . Divergent roles for Fc receptors and complement in vivo . Ann Rev Immunol 1998 ; 16 : 421432 .
  • 55
    Qian Z , Jakobs FM , Pfaff-Amesse T , Sanfilippo F , Baldwin WM III . Complement contributes to the rejection of complete and Class I MHC incompatible cardiac allografts . J Heart Lung Transplant 1998 ; 17 : 470478 .
  • 56
    Nakashima S , Qian Z , Rahimi S , Wasowska BA , Baldwin WM III . Membrane attack complex contributes to destruction of vascular integrity in acute lung allograft rejection . J Immunol 2002 ; 169 : 46204627 .
  • 57
    Selvan RS , Kapadia HB , Platt JL . Complement-induced expression of chemokine genes in endothelium: regulation by IL-1-dependent and -independent mechanisms . J Immunol 1998 ; 161 : 43884395 .
  • 58
    Saadi S , Holzknecht RA , Patte CP , Platt JL . Endothelial cell activation by pore-forming structures: pivotal role for interleukin-1alpha . Circulation 2000 ; 101 : 18671873 .
  • 59
    Ey PL , Prowse SJ , Jenkin CR . Complement-fixing IgG1 constitutes a new subclass of mouse IgG . Nature 1979 ; 281 : 492493 .
  • 60
    Ey PL , Russell-Jones GJ , Jenkin CR . Isotypes of mouse IgGI. Evidence for ‘non-complement-fixing IgG1 antibodies and characterization of their capacity to interfere with IgG2 sensitization of target red blood cells for lysis by complement . Mol Immunol 1980 ; 17 : 699710 .
  • 61
    Neuberger MS , Rajewsky K . Activation of mouse complement by monoclonal mouse antibodies . Eur J Immunol 1981 ; 11 : 10121016 .
  • 62
    White KD , Frank MB , Foundling S , Waxman FJ . Effect of immunoglobulin variable region structure on C3b and C4b deposition . Mol Immunol 1996 ; 33 : 759768 .
  • 63
    Yokoyama I , Waxman F . Differential susceptibility of immune complexes to release from the erythrocyte CR1 receptor by factor I . Mol Immunol 1994 ; 31 : 227240 .
  • 64
    Bindon CI , Hale G , Waldmann H . Importance of antigen specificity for complement-mediated lysis by monoclonal antibodies . Eur J Immunol 1988 ; 18 : 15071514 .