This approach is claimed to work by inducing disease via the idiotypic network. The use of an autoantibody (Ab1) with a theoretically pathogenic idiotype as the immunizing agent leads to the generation of an anti autoantibody (Ab2; anti-idiotype) by the immunized animal. Subsequently, the animal develops an anti-anti-autoantibody (Ab3; anti-anti-idiotype) which may have similar binding characteristics to the original immunizing autoantibody (Ab1; idiotype). However, the precise relationship between the idiotypic circuitry and the clinical expression of the obstetric complications of APS is obscure.
Researchers have mainly immunized naive mice (such as BALB/c mice) with murine or human aCL, and in some instances, with antiphosphatidylserine antibodies (aPS) (Yodfat et al 1996) or anti-DNA antibodies (Blank et al 1992). Successful reports of APS induction, including autoantibody (aPL) production, have been reported by Bakimer et al (1992, 1993 & 1995), Blank
et al (1992), Cohen et al (1993 & 1994) and Sthoeger et al (1993a & b). The studies have demonstrated the pathogenic potential (pp) of aCL (IgG higher pp than IgM) to induce a form of APS in BALB/c mice. Common clinical features of induced APS in all these studies were the poor outcomes of pregnancies relating to decreased embryo and placental weights, increased foetal resorptions and low pregnancy rates. Other shared features include prolonged activated partial thromboplastin time (pAPTT) and
thrombocytopenia (Sthoeger et al 1993a & b, Bakimer et al 1993, Cohen et al 1993 & 1994).
Sthoeger et al (1993a) induced APS with a mouse monoclonal IgM aCL (20402), which was shown to impair the implantation phase of embryonic development. Specific binding of this aOL to the trophectoderm cell lineage of
in vitro implanted normal embryos was observed.
The study by Yodfat et ai (1996) successfully induced APS which they claimed was through the idiotypic network in BALB/c mice, using another antiphospholipid antibody, aPS, affinity purified from patients with APS. Mice were immunized with human IgM and IgG aPS, but only those immunized with IgG a PS developed high titres of mouse a PS and clinical parameters of APS (pAPTT, thrombocytopenia and increased foetal resorption rate).
BALB/c mice have also been immunized with a monoclonal IgM anti-dsDNA antibody (MIV-7) which carries the anti-DNA antibody 16/6 idiotype, resulting in APS and SLE (Blank et al 1992). The mice produced IgG aCL, LA (characterized by pAPTT) and developed thrombocytopenia and poor pregnancy outcome (high resorption rate and low placental / foetal weights), indicating their affliction with APS. When mice were immunized with IgG aCL, APS was induced without SLE.
In Fishman et al’s report, in vitro cytokine production by spleen cells derived from IgM aCL (H-3) immunized BALB/c mice was measured (Fishman et al 1992). Decreased levels of Interleukin-2 (IL-2), IL-3 and Granulocyte- macrophage colony-stimulating factor (GM-CSF) were detected, produced by splenocytes of mice with induced APS. The serum IL-3 level is lower in pregnant patients with APS compared to control serum. IL-3 and GM-CSF have been shown to increase the chances of foetal survival when injected into abortion-prone mice (CBA/J x DBA/2), reducing resorption rates (Chaouat et al 1990). Thus, the decreased fecundity rate and high resorption rates observed in this study may be directly linked to the decreased cytokine production in the mice with induced APS.
A human monoclonal IgM aCL (H-3) has been used to immunize BALB/c mice actively. Upon developing experimental APS, pregnant mice were i.v. infused with either ox-LDL (oxidized low-density lipoprotein), native LDL or phosphate-buffered saline (PBS) (George et al 1997). The clinical picture of APS (thrombocytopenia, higher foetal resorption rate and prolonged APTT) in mice administered ox-LDL was displayed in a more severe form compared to mice infused with native LDL or PBS. Conclusively, ox-LDL, compared with native LDL, aggravates some clinical manifestations of experimental APS (George et al 1997). This may be explained by the reactivity of ox-LDL with phospholipids.
Experimental APS, induced by H-3, in female BALB/c mice also manifested itself neurologically (Ziporen et al 1997). The mice performed less accurately compared to controls in placing reflex, postural reflex and grip tests, pointing to affected CNS function in the cortex level and brain stem. Spatial behaviour, motor co-ordination and learning ability were also impaired. The authors suggest the combination of elevated aPL, anti-endothelial cell
antibodies and anti-p2GPI antibody levels have some involvement in the
neurological and behavioural defects shown. The autoantibodies may bind directly to the neurons, or may interfere with haemostasis, leading to occlusion of microvessels in the basal ganglia.
If therapeutic strategies are to be determined for the treatment of neurological, coronary and thromboembolic complications associated with human APS, the animal models need to be ‘fine-tuned’ to produce these “second hits” more often. It has become apparent that some “trigger” other than the presence of aCL or LA after active immunisation is required to produce these symptoms of APS.
Active Induction with an autoantigen (P2GPI)
Immunisation of NIH / Swiss mice with P2GPI in Freund’s complete adjuvant
(FCA) resulted in production of two non-cross-reactive antibody populations;
anti-p2GPI and aPL antibodies (Gharavi et al 1992). Pierangeli and Harris
(1993) confirmed these results in BALB/c mice, using lipid-free adjuvants to
anti-p2GPI antibodies, indicating that the aCL were not being induced via the
idiotypic network, if this network were in operation, the anti-P2GPI antibodies
would have appeared first. This suggests that proteins (such as P2GPI) with
high avidity for phospholipids (which are poor immunogens) transform these phospholipids into immunogens, and induce aPL.
Neurological function and brain pathology of BALB/c mice immunized with
P2GPI were compared with control mice immunized with bovine serum
albumin (BSA) (Hess et al 1994). Despite the presence of aCL, the P2GPI-
immunized mice showed no evidence of neurological impairment or cerebral infarction when compared to the controls.
Naive BALB/c mice were immunized with P2GPI, resulting in elevated levels
of aCL, aPS, aPI and anti-P2GPI antibodies (Blank et al 1994b).
Subsequently, pAPTT, thrombocytopenia and high foetal resorption rates were observed, indicating that the antibodies induced were pathogenic and played a key role in the development of experimental APS. However, Silver
et al (1995) having also immunized BALB/c mice with P2GPI [albeit with
methodological diferences eg. lOug, intradermally (Blank et al 1994b) versus lOOug, subcutaneously (Silver et al 1995)] reported that in spite of the
antibody production (aCL and anti-p2GPI antibodies), the clinical findings did
not differ significantly from control mice. This study implies that at least some aCL are not pathogenic and is consistant with findings in humans where
antibody titre does not always correlate with clinical activity (Qamar et al 1990).
The thrombogenic effect of murine aCL has been demonstrated by Pierangeli
et al (1996), using a unique mouse model that enables the study of the kinetics of formation and disappearance of a thrombus induced in the femoral veins of the study animals (Pierangeli & Harris 1994, Pierangeli et al 1994 &
1995). CD1 mice were actively immunized with P2GPI (group 1) and purified
human IgG (group 2). Group 1 mice produced aCL and anti-P2GPI
antibodies, while group 2 mice produced murine aCL and anti-human IgG antibodies. The mean thrombus area as well as mean time during which thrombi persisted were significantly greater in group 1 and 2 mice compared with control mice which had been immunized with human serum albumin (HSA). There was no statistical difference between groups 1 or 2, showing
that there was no influence of anti-p2GPI antibodies (groupl) on thrombus
formation, compared with mice producing aCL alone (group2). The aCL induced in this study have the same thrombogenic effect previously demonstrated in similar mice passively immunized with human aCL (Pierangeli & Harris 1994, Pierangel et al 1994). Therefore, thrombogenic effects are related to aCL specificity and are not a function of the source of aCL. In this “Pierangeli model”, the methodology may restrict the demonstration of any other symptoms of APS in the mice.
A more recent study showed that all p2GPI-immunized PL/J mice developed
aCL, anti-DNA antibodies and ANA (anti-nuclear antibodies), and significantly smaller litter size (compared to the immunized and non-immunized controls) (Garcia et al 1997). No thrombocytopenia was evident and may have needed a longer follow-up to develop (the mice were checked at 14 weeks of age). However, as part of a long-term study, four of the mice were injected with
P2GPI-induced aPL, to determine the natural course of APS. After eight
months, two of the four mice showed progressive neurological symptoms with flaccid paralysis and trophic changes in the tail and hind limbs. One of these two had a large thrombus in the thoracic spine Qust below the neck) which was compressing the muscle, nerves and spinal cord. Vacuolization and degeneration of spinal cord neurones, and thrombosis in small vessels was observed. After 10 months, the third animal developed spastic paralysis
of both hind limbs. P2GPI -induced aPL are pathogenic in this mouse model,
and cause foetal growth retardation and thrombosis. This study is the first
description of neurological involvement and thrombosis in mice with P2GPI-
induced APS (Garcia et al 1997).
Although the immunogen responsible for aPL in autoimmune diseases has
not been identified, it is clear that immunisation of mice with P2GPI results in
antibodies that have the same characteristics as those detected in autoimmune disease.
Active induction with other antigens Svnthetic peptides
NIH/Swiss mice have been immunized with synthetic peptides (which share
structural homology with the PL-binding sites of P2GPI) conjugated to BSA in
Freund’s adjuvant (Gharavi et al 1997). Animals immunized with one of these peptides produced significantly higher levels of aPL antibodies compared to controls. The peptides used spanned portions of known viral proteins, and this study was based on the hypothesis that PL-binding proteins, such as viral proteins, may induce aPL production. The results demonstrate that PL-
binding viral proteins may induce aPL production similar to P2GPI, and may
be responsible for the induction of aPL production in patients with APS.
Oestrogen
A non-autoimmune mouse strain, C57BL/6J, has been shown to spontaneously express low levels of autoantibodies to CL and dsDNA (Ansar Ahmed & Verthelyi 1993). When these mice were treated with oestrogen (administered by subcutaneous pellet) and no other autoantigen, autoantibody levels were augmented. Persistent detectable levels of IgG and IgM aCL were induced. This effect of oestrogen on the immune system persisted for months after the exposure to exogenous oestrogen had ended (Verthelyi & Ansar Ahmed 1997). Anticardiolipin binding was not dependent
upon the presence of P2GPI. Addition of exogenous human P2GPI reduced,
epitope(s) recognised by the murine aCL antibodies could be partially
blocked by the presence of human P2GPI (Verthelyi & Ansar Ahmed 1997).