CD88 was the first identified receptor for the complement activation fragment C5a, a potent immune and inflammatory mediator. Recent work from our lab, suggests that signaling through CD88 may play a detrimental neuroinflammatory role in AD. After defining the specificity of a monoclonal rat anti-mouse CD88 clone 10/92 antibody using CNS derived cells from wild type and CD88-/- mice, we demonstrated increased expression of the receptor primarily in the vicinity of thioflavine positive Aβ plaques in murine transgenic models of AD. CD88 immunoreactivity was associated with Iba-1 and CD45 positive cells, indicative of microglia, and localized predominantly to the processes of plaque associated microglia and polarized toward the Aβ plaque. These observations are consistent with the proposed chemotactic response to C5a and the potential for costimulation of CD88 and receptors for plaque associated components (Zhang et al. 2007
;Walter et al. 2007
;Jana et al. 2008
). It was also determined that in mice treated with the specific CD88 antagonist, PMX205, microglia surrounding plaques continue to express CD88 without noticeable up- or down-regulation of receptor density. Finally, no alterations in C1q association with plaques or C3 induction in astrocytes were detected after prolonged treatment with PMX205, suggesting that any beneficial functions of these early complement components may remain undisturbed due to their continued production during treatment with the CD88 antagonist.
Thus far, there have been conflicting results reported regarding CD88 expression in the AD brain. O’Barr et al, using a polyclonal rabbit anti-human CD88 antibody, directed against the N-terminus of CD88, reported comparable CD88 expression in neuronal cell bodies in both normal and AD human brain (O’Barr et al. 2001
). In contrast, using both rabbit polyclonal and mouse monoclonal anti-human CD88 antibodies, Farkas and colleagues reported that neuronal CD88 expression was reduced in the human AD brain (Farkas et al. 2003
). The reduction was restricted to neurons, and observed in a broad range of brain areas, although reactivity was concentrated around Aβ plaques (at the time hypothesized to be due to dystrophic neuritis, not microglia). No analysis of mRNA was reported in this study. Similar expression of CD88 protein was reported in non-transgenic and Tg2576 mice, using a polyclonal rabbit anti-mouse CD88 antibody directed against the N-terminus, and neuronal CD88 mRNA expression was also reported using in situ hybridization analysis in wild type mice (O’Barr et al. 2001
). During our initial experiments comparing CD88 protein levels in Tg2576, Arc48 mice, and non-transgenic mice using a polyclonal rabbit anti-mouse CD88 antibody also developed against the N-terminus of murine CD88 (C1150-32, BD Pharmingen), we observed an increase in neuronal CD88 in the Tg2576 mice, relative to our non-transgenic controls (data not shown). However, control analysis in CD88-/- brain tissue revealed a similar neuronal staining pattern to that found in our non-transgenic mice. Thus, no precise conclusion as to the identity of the increased staining with the C1150-32 antibody in our Tg2576 mice could be made. Reports of other inconsistencies in the protein expression pattern for CD88 detected with both polyclonal and monoclonal antibodies directed against the N-terminus, have been previously noted (Kiafard et al. 2007
). In that study, the authors present data demonstrating no antibody reactivity in mouse renal tubular cells using clone 10/92, which had previously been reported to express CD88 using a polyclonal anti-CD88 antibody (Bao et al. 2005
). Cells of myeloid lineage, such as macrophages, however, were found to express CD88 using the clone 10/92 antibody. While the above reports did not validate the specificity of their staining using CD88-/- mice, in our study here, the specificity of the monoclonal anti-CD88 clone 10/92 antibody was demonstrated by positive reactivity in flow cytometric, western blot analysis and immunocytochemistry on murine primary microglia cells derived from wild type mice, but lacking in microglia derived from CD88-/- mice. Using the clone 10/92 we also showed intense staining on microglia in the area of fAβ plaques in AD mouse models, in line with previous reports demonstrating antibody reactivity in cells of myeloid lineage (Kiafard et al. 2007
;Tschernig et al. 2007
). Further evidence for microglial CD88 protein expression, the C1150-32 anti-CD88 antibody was also able to distinguish between wild type and CD88-/- microglial CD88 expression by flow cytometric and Western blot analysis, but had no reactivity in immunocytochemistry. RT-PCR analysis, verified that our cultured microglia were also expressing CD88 mRNA, which was absent in CD88-/- microglia and in both wild type and CD88-/- immature and mature cultured cortical neurons. One explanation for the lack of CD88 mRNA observed in our neuronal cultures, in contrast to that shown by others, could be a lack of extracellular signals, perhaps coming from surrounding glia, necessary for CD88 mRNA expression. Although mRNA for CD88 has been demonstrated in neurons in vivo
, the levels observed in the absence of CNS insult, have been generally very low (Paradisis et al. 1998
;Stahel et al. 1997
). We did observe increased CD88 mRNA in our AD mice (data not shown) using RT-PCR of whole brain extracts, however, in situ
hybridization will be necessary to determine if neurons contribute to the increase in mRNA in these animal model of neurodegeneration. Lack of specificity of antibodies against another CNS receptor (a7nAcR) was also observed using knock out mice (Moser et al. 2007
;Herber et al. 2004
), demonstrating that this situation is not unique to CD88 and stresses the importance of validating immunoreactivity results using knockout animals where possible.
Previous studies have shown that recombinant human C5a is chemotactic for both microglia and astrocytes in vitro
(Yao et al. 1990
). In this study clone 10/92 anti-CD88 antibody did not immunolabel astrocytes in either wild type or AD mice. This is surprising given our previous studies in a rat model of amyotrophic lateral sclerosis, which showed strong expression of CD88 on astrocytes from SOD1G93A
transgenic rats using an anti-rat CD88 monoclonal antibody (Woodruff et al. 2008
) as well as the results of others demonstrating astrocyte expression of CD88 (Gasque et al. 1997
;Lacy et al. 1995
). The lack of anti-CD88 reactivity on astrocytes (as well as neurons, in culture) could be a difference in conformation of the receptor between those cell types and microglia, although there have been no reported cell specific differences in posttranslational modification of CD88. Alternatively, since CD88 has been reported to associate with other receptors, astrocytes could lack a co-receptor that may associate with CD88 in microglia or express a surface molecule that masks the CD88 epitope recognized by the clone 10/92 anti-CD88 antibody. These results taken together with the existence of a second C5a receptor, C5L2, suggest that caution is needed when using antibody reactivity to assess CD88 protein levels in the murine CNS or interpreting previous reports based on antibody reactivity or C5a-induced responses. Alternative detection methods, such as gene silencing or in situ hybridization, are necessary unless specificity of the antibody being used is demonstrated using CD88-/- tissue or cells. Furthermore, additional experimental approaches (novel specific anti-CD88 antibodies) are needed to further establish CD88 expression in AD models.
Previously, we reported that the administration of PMX205 resulted in reduced GFAP and CD45 reactivity (markers for activated astrocytes and microglia, respectively) as well as decreased plaque and insoluble Aβ42 loads in two distinct AD mouse models (Fonseca et al. 2009
). The cyclic hexapeptide C5a receptor antagonist was administered at an age at which fAβ deposition is known to occur, and thus, at a time when C5a is likely generated by complement activation. The initial interaction of C5a with CD88 and Aβ with TLR2 (or 4) could then synergistically induce TNF-α (Jana et al. 2008
), which can be directly neurotoxic, and may also upregulate CD88 on glia. This enhanced CD88 signaling would lead to greater TNF-α production and thus acceleration of pathology. However, other possibilities exist. For example, if a form of CD88 is upregulated on neurons as a result of the accumulating injury; PMX205 may also be directly neuroprotective (Farkas et al. 1998
) and/or result in decreased amyloid peptide production or tau phosphorylation which would prevent/slow the progression of cognitive decline. Further study will be needed to resolve the precise mechanism of PMX205 action in this and other neurodegenerative diseases.
In any event, the specific inhibition of CD88 signaling would nevertheless allow the progression of other previously demonstrated and hypothesized protective effects of complement activation up-stream of C5a generation (Pisalyaput and Tenner 2008
;Wyss-Coray et al. 2002
;Maier et al. 2008
). When in complex with the proenzymes C1r and C1s in C1, C1q binds fAβ and thus activates the classical complement cascade (Rogers et al. 1992
), which can in turn generate C5a. However, C1q alone has been demonstrated to have multiple effects in other disease states, including the ability to enhance phagocytosis of apoptotic cells (Botto et al. 1998
) and suppress proinflammatory cytokines (Fraser et al. 2009
) and the ability to provide direct protection to injured neurons (Pisalyaput and Tenner 2008
). Since C1q is associated with thioflavine positive fAβ plaques even following PMX205 treatment, our detection of its continued association with plaques in the presence of PMX205 demonstrates that it is possible that C1q may continue to play a protective role in AD by mediating clearance of cellular debris and preventing neuronal damage. Similarly, it has been suggested that C3 cleavage products may facilitate removal of aggregated Aβ, as inhibition of C3 cleavage through the over-expression of the complement receptor related protein y (Crry) or complete C3 genetic deletion in AD mouse models resulted in a substantial increases in Aβ deposition (Maier et al. 2008
;Wyss-Coray et al. 2002
). In this study, we found that C3 production by astrocytes, which is elevated in AD mouse models (Zhou et al. 2008
), is not substantially altered relative to the thioflavine plaque load with PMX205 treatment, and therefore, C3 as well as C1q may continue to provide neuroprotective functions during PMX205 treatment.
In summary, we have observed microglial upregulation of CD88 in response to amyloid deposition, a localization of receptor reactivity in microglia surrounding Aβ plaques and polarization of the receptor to microglial processes in closest proximity to the plaques. In addition, our data further supports the targeting of the anaphylatoxin receptor CD88 as a therapeutic approach to the treatment of AD, since detrimental activities are inhibited, while potentially protective activities of complement are preserved.