Despite multiple studies of anti-Aβ immunotherapy in mice, there is still no consensus on how anti-Aβ immunotherapy works [14, 15], particularly as it relates to the role of microglial activation. It was originally proposed that Aβ immunization triggers phagocytosis of antibody-bound Aβ immune complexes via microglial FcR. After immunization, increased number of microglial cells stained with anti-Aβ antibodies were observed . Indeed, using an ex vivo strategy, it was shown that anti-Aβ antibodies induce phagocytosis of Aβ plaques . Importantly, Fab fragments of these antibodies fail to induce Aβ phagocytosis, suggesting that the enhanced uptake is attributable to FcR . Subsequent studies have shown that at least in Tg2576 APP mice, a role for enhanced phagocytosis of mAb:Aβ complexes via the FcR can largely be ruled out, since Aβ1-42 immunization in Tg2576 × FcRγ-/- crossed mice was effective in reducing Aβ loads . Additional studies now show that an intact mAb (and therefore FCR interactions) is not required for efficacy; since Fab fragments  and scFv fragments (Levites and Golde, unpublished observation) are efficacious in immunotherapy. Several groups have reported that following Aβ immunotherapy, there are transient or stable enhancements of microglial activation associated with Aβ removal; whereas others do not find this [1, 21–23]. Furthermore, in humans receiving the AN-1792 vaccine, Aβ-laden microglia have been noted in postmortem studies . Although antibody and microglial Fc receptor-mediated interactions have been suggested to activate microglia following vaccinations, other inflammatory consequences may play a role in this paradigm. Based on published reports, it has been suggested that clearance of amyloid deposits in patients enrolled in the AN-1792 trial may have been due to an adverse inflammatory response to the vaccine rather than due to the anti-Aβ antibodies . This proposition may be supported by some recent reports, wherein induction of experimental autoimmune encephalitis (EAE) and nasal vaccination with glatiramer acetate reportedly decrease amyloid plaques in APP transgenic mice . Another report by the same group shows that, in mice over expressing IFN-gamma in the CNS, amyloid vaccination lead to meningoencephalitis and T cell-dependent clearance of amyloid plaques from the brain . Both of these reports provide evidence that peripheral inflammatory responses and CNS autoreactive T cells may play a role in vaccination-induced clearance of plaques. Furthermore, some recent reports have indicated that inflammatory insults, either by injecting LPS directly into the brain [44, 50] or overexpression of TGF-β in the CNS , can result in reductions of amyloid deposits. Enhanced microglial activation was noted in both of these reports and is suggested to contribute to the clearance of amyloid deposits.
In this report, we sought to determine the role of IL-1-mediated microglial activation on IL-1-mediated inflammatory responses following Aβ vaccination and on Aβ deposition during normal aging using interleukin-1 receptor 1-knockout (IL-1 R1-/-) mice [40–42] that were crossed to APP Tg2576 transgenic mice (APP/IL-1 R1-/-). We first tested the efficacy of Aβ immunization in APP/IL-1 R1-/- mice. Our results show that passive immunization with an anti-Aβ mAb is effective in reducing plaque loads both in APP/IL-1 R1-/- mice and APP/IL-1 R1+/- littermates, when immunization is started prior to significant plaque deposition. However, as we have seen previously, immunization was not efficacious in mice that have pre-existing Aβ loads [17, 18, 52]. Thus, these results support our general hypothesis that microglial activation may not be required for efficacy of immunization in Tg2576 mice. The lack of IL-1 R1 (in -/- mice) did not significantly alter Aβ deposition in untreated mice. There were no significant differences in total extractable Aβ levels or overall histochemical loads, at any time, between the APP/IL-1 R1-/- mice and APP/IL-1 R1+/- littermates compared to wild type Tg2576 mice (IL-1 R1+/+). Curiously, in 2 of 7 15-month-old APP/IL-1 R1-/- mice examined, an unusual pattern of Aβ plaque staining was noted, with an abundance of diffuse immuno-reactive Aβ plaques in the neocortex of these mice. It is not clear at this time whether this unusual pattern of diffuse Aβ deposits is due to the IL-1 R1-/- phenotype or some mouse background effect. We then examined the effects of IL-1 R1 knockout on the state of microglial activation and astrogliosis surrounding amyloid plaques deposits. For microglial staining, we used two well characterized markers for microglial activation, anti-mouse CD45 and Iba1, and for activated astrocytes we used anti-GFAP staining. Our results show that there were abundant numbers of CD45 and Iba1 immuno-reactive microglia present, surrounding Aβ plaques in APP/IL-1 R1-/-, APP/IL-1 R1+/- and wild type Tg2576 mice (IL-1 R1+/+), with no significant differences in the immuno-reactivity of staining using these markers. Similarly, robust GFAP staining was seen in all three groups of mice analyzed, with no significant differences seen in the GFAP immuno-reactivity comparing all three groups of mice.
Based on our immuno-staining analysis, we were not able to ascertain whether abrogated IL-1 signaling in the IL-1 R1-/- mice blunted the inflammatory microglial response or astrogliosis in the region of deposited Aβ plaques. Previous experiments in IL-1 R1-/- mice have shown abrogated IL-1-mediated responses following acute inflammatory stimuli. In a stab wound model of injury in the brain, IL1-R1-/- mice had fewer amoeboid microglia/macrophages near the sites of injury, mildly abrogated astrogliosis and reduced expression of cytokines induced by IL-1 expression . In another report, IL-1 R1-/- mice had reduced IL-6 and E-selectin expression, and reduced IL-1-induced fever and acute phase responses to turpentine . However, IL-1 R1-/- mice do not differ from control mice in their responses to either a lethal challenge with D-galactosamine plus LPS or high dose LPS , indicating that IL-1 R1 signaling functions may not be necessary for the response to LPS. Thus, it is possible that the chronic nature of the microglial response during the course of amyloid deposition may abrogate any acute or subtle signaling events mediated through the IL-1 R1 receptor. Certainly, it is possible that other receptors for IL-1 may compensate for the lack for IL-1 R1 in this situation. Besides the IL-1 R1 and IL-1 RII receptors, the recently reported P2X7 receptor has also been implicated to be a key player in IL-1 signaling  and could compensate for the lack of IL-1R1 in IL-1 mediated signaling events. Alternatively, the microglial response to deposited Aβ may not require signaling through the IL-1 R1 receptor. The LPS receptor (CD14) [54, 55], the scavenger receptor complex (CD36)  and toll-like receptors (TLR-2, TLR-4)  can directly activate microglia in response to amyloid deposition, possibly circumventing any IL-1 R1-mediated signaling events in the IL-1 R1-/- mice.
Like our previous studies, these studies suggest that microglial activation is not required for immunization to work in Tg2576 mice, although this should not be viewed as definitive. As indicated above, in the IL-1 R1-/- mice, microgliosis and astrogliosis are mildly abrogated at best and do not result in microglial paralysis. Thus experiments using recently developed CD11b-HSVTK mice. developed by Aguzzi and colleagues . that enable selective killing of microglia cells may provide more definitive results.