Our results confirm earlier findings demonstrating profound mechanical allodynia resulting from intravenous ch14.18 treatment, an antibody that has recently been shown to improve the outcome of patients with high risk neuroblastoma in a phase III clinical trial. Our principle novel finding is that magnitude and especially duration of ch14.18-induced mechanical sensitization are due in great part to the ability of this antibody to fix complement. Animals treated with an antibody variant with reduced capacity to fix complement (hu14.18K322A), displayed a tendency towards reduced allodynia for the first 2 h post-injection. More importantly there was a much faster recovery from allodynia back to basal mechanical withdrawal threshold values; differences were readily apparent just a few hours after injection. This decrease occurred despite the fact that hu14.18K322A retained ADCC activity.
The complement system consists of serum and membrane bound proteins that interact with each other and with immune cell molecules in a stylized cascade and serves to amplify the original signal. Many complement proteins act as receptor-specific activator molecules [1
]. Although there are three major complement pathways, the classical pathway activated by antigen–antibody complexes, is most likely to be activated by anti-GD2
antibody. This results in formation of complement factors C3a and C5a as well as membrane attack complex (MAC), which consists of complement factors C5b joined to C6–C9. The macromolecule thus formed is also known as terminal complement complex.
Complement activation is acknowledged to be a major factor in several systemic inflammatory disorders and autoimmune diseases such as reperfusion injury, rheumatoid arthritis and Guillain Barré Syndrome [21
]. There is also a precedent for involvement of complement in local inflammation generated by antibody-antigen reactions and pain. Complement factors C1, C1q, C3, C3d and C9 are released into the spinal dorsal horn following nerve injury [25
] and, although pain behavior was not measured in these studies, it is likely to have occurred as the degree of localized spinal glial activation generated in these experiments is frequently associated with spinal sensitization and hyperalgesia. More recently, three models of neuropathic pain were all shown to induce increases in mRNA for complement factors C1q, C3, C4 and C5 specificallyin microglia [16
]. Increases in complement factor mRNA peaked 3–7 days after the nerve injury.
Complement receptor 1 is a high affinity receptor for C3b and C4b, it regulates complement activation and prevents activation of C3 and C5 convertase, thus, blocking formation of complement components C3a and C5a as well as membrane attack complex [1
]. Spinal administration of the antagonist, soluble complement receptor 1, totally reverses already established mechanical allodynia due to neuroinflammation or to nerve injury [34
]. It is proposed that this reversal was due to blockade of MAC formation, although actions through complement factor C5a are also likely. Our data support their conclusion that, complement fixation, followed by generation of both C5a and MAC, is involved in neuropathic pain.
In our study, animals pretreated with a complement factor C5a receptor antagonist displayed no mechanical allodynia throughout the experiment. This contrasts with our observation that hu14.18K322A injection, with limited complement activation, resulted in allodynia for the first 2 h. This difference between animals given the C5a receptor antagonist and those given the mutated antibody must be due to the residual complement activation of the hu14.18K322A and its actions through generation of C5a and point to a unique role for this factor in initiating the pain state. In the periphery, intradermal injection of the C5a complement fragment, elicits mechanical hyperalgesia peaking 20 min after injection [23
]. Complement fragment C5a, as well as the other anaphylatoxic peptide C3a, induce the release of histamine and other inflammatory mediators from mast cells [20
]. However, previous experiments in our laboratory using rats pretreated for several days with compound 48/80, a potent degranulator of mast cells, followed by administration of ch14.18, resulted in only a trend towards reduction in the magnitude of mechanical allodynia as compared to rats with control pre-treatment, and no change in duration of the pain behavior (Sorkin and Yu, unpublished results). These data indicate that mast cell degranulation, at best, contributes only a small component of the observed ch14.18-evoked allodynia. Nanomolar concentrations of complement-derived C5a have a chemotactic effect on neutrophils [30
] and other inflammatory cells, and they also induce upregulation of adhesion molecules and cause increased vascular permeability [21
]. Subsequent activation of infiltrating immune cells results in release of oxygen free radicals and lysosomal proteases. Peripheral blockade of the C5a receptor by systemic administration of a receptor-specific antagonist reduces, but importantly does not totally block induction of, mechanical sensitization associated with paw incision. Antagonist pre-treatment also reduces acute local production of several pro-inflammatory cytokines [8
]. These data further support the hypothesis that C5a contributes to induction and maintenance of pain behavior.
While there is no doubt that C5a elicits peripheral inflammation, infiltration of neutrophils and hyperalgesia on its own [23
], that is probably not the only complement-dependent mechanism by which binding of ch14.18 to GD2
ganglioside along the nerve trunk causes allodynia and ectopic activity. In these experiments, we observed that lack of C6 in the PVG (C−) animals, downstream of presumably intact C5a formation, is sufficient to significantly reduce the anti-GD2
-induced pain behavior in the induction phase (0–2 h) and particularly in the later portions (after 2 h) of the observation period. The most likely mechanism of this reduction was loss of MAC formation. This loss was less complete than that seen following pre-treatment with C5a receptor antagonist. Injection of hu14.18K322A into C6 deficient animals had a strong tendency to result in less allodynia than did injection of ch14.18, although this was significant only for the lower dose during the first post-injection period (0–2 h). As the difference between these two antibodies is their ability to fix complement and both sets of animals were unable to form MAC, the disparity must be due to complement mediated factor upstream of C6, most likely complement factor C5a.
Insertion of MAC into cell membranes results in the formation of transmembrane pores. Depending on MAC concentration, potential outcomes range from calcium influx and excitation (activation) to cell lysis and death [19
]. Sublytic levels of MAC produce protein kinase C activation, production of diacylglycerol, ceramide and arachidonic acid metabolites as well as activation of mitogen-activated kinase pathways in various cell types [19
]. Recently, Koski and colleagues [9
] demonstrated that MAC induces increase in Jun N-ternial Kinase (JNK) activity in Schwann cell cultures within a time course roughly corresponding to onset of ch14.18-evoked pain behavior. Membrane attack complex is found in serum and cerebrospinal fluid and on Schwann cells in rats with experimental allergic neuritis [18
] as well as in patients with Guillain–Barré syndrome. In these patients, presence of MAC correlates with progression of clinical signs [28
]. In animals with experimental allergic neuritis, inhibition of complement activation delays and or totally suppress demyelination and disease onset [10
An interesting study from Anderson and colleagues indicates that C6 deficient rats exhibit milder motor deficits and faster recovery than wild type controls following spinal cord contusion, suggesting that MAC contributes to spinal cord excitotoxicity [13
]. These animals were not tested for pain behavior.
However, results from Costigan and Woolf’s group show that nerve injury-elicited pain behavior was dependent, in part, on C5a activation in the spinal cord and not on factors including or down stream of C6 [16
]. The lack of MAC involvement was thoroughly demonstrated using C6 deficient rats similar to those that we obtained, and by a reversal of allodynia with intrathecal administration of the C5a receptor antagonist. There are several important differences between the two studies. We examined responses to innocuous mechanical pressure within hours of the insult. Indeed, maximal allodynia was achieved within 30–45 min. Woolf examined cold allodynia and hyperalgesia in response to pinprick [16
] 3 days post injury. The injuries, although both clinically relevant, are quite different with respect to time of onset and reversibility. Overt surgical injury to a nerve vs. administration of an antibody that acts along the axon to elicit ectopic activity [36
] and is likely to predominantly activate the classical complement pathway. Anti-GD2
induced-pain is completely resolved in children within 2–24 h after completion of treatment [35
]. Thus, it should not be surprising if the mechanisms differ, despite complement involvement in both.
In summary, our findings demonstrated that the mutant hu14.18K322A displayed greatly reduced capacity to mediate CDC while retaining significant ADCC activity, as compared to ch14.18. In animal studies, the first set of experiments showed that anti-GD2 induced allodynia is reduced by use of an antibody with a point mutation that reduces complement activation. Similar reductions in allodynia were observed following injection of the original antibody in an animal with deficient C6 complement, a deficit that would prevent the formation of MAC. Complete abrogation of allodynia was achieved with antagonism of complement receptor C5a. Thus, we propose that complement activation, including formation of both C5a and MAC are components of anti-GD2 antibody-induced allodynia.