AA-amyloidosis can be transferred by peripheral blood monocytes

Abstract

Spongiform encephalopathies have been reported to be transmitted by blood transfusion even prior to the clinical onset. Experimental AA-amyloidosis shows similarities with prion disease and amyloid-containing organ-extracts can prime a recipient for the disease. In this systemic form of amyloidosis N-terminal fragments of the acute-phase reactant apolipoprotein serum amyloid A are the main amyloid protein. Initial amyloid deposits appear in the perifollicular region of the spleen, followed by deposits in the liver. We used the established murine model and induced AA-amyloidosis in NMRI mice by intravenous injections of purified amyloid fibrils ('amyloid enhancing factor') combined with inflammatory challenge (silver nitrate subcutaneously). Blood plasma and peripheral blood monocytes were isolated, sonicated and re-injected into new recipients followed by an inflammatory challenge during a three week period. When the animals were sacrificed presence of amyloid was analyzed in spleen sections after Congo red staining. Our result shows that some of the peripheral blood monocytes, isolated from animals with detectable amyloid, contained amyloid-seed that primed for AA-amyloid. The seeding material seems to have been phagocytosed by the cells since the AA-precursor (SAA1) was found not be expressed by the monocytes. Plasma recovered from mice with AA amyloidosis lacked seeding capacity. Amyloid enhancing activity can reside in monocytes recovered from mice with AA-amyloidosis and in a prion-like way trigger amyloid formation in conjunction with an inflammatory disorder. Human AA-amyloidosis resembles the murine form and every individual is expected to be exposed to conditions that initiate production of the acute-phase reactant. The monocyte-transfer mechanism should be eligible for the human disease and we point out blood transfusion as a putative route for transfer of amyloidosis.

Figure 1. Spleen amyloid deposits stained with Congo red.

(A) The amyloid appears pink and is localized to the perifollicular zone. (B) The identical area exhibits green birefringence in polarized light. Amyloid is indicated by arrows.

Figure 2

(A) Flow cytometry analysis of isolated and cultured fraction of peripheral blood monocytic cells (PBMC). The measurements were performed using forward scatter versus side scatter and 10,000 events were recorded. The marked area represents monocytic population and four independent isolations were analyzed. The monocyte population was determined to 64%±15% (mean±SD). Insert shows analysis of PBMC prior to culture. (B) Representative picture of a cell recovered after isolation and culture. Bar 1 uM.

Figure 3. Analysis of AA/SAA reactivity in peripheral blood monocytes by confocal microscopy showed immunoreactivity in 5% of the monocytes isolated from a mouse with AA-amyloidosis (A).

There was no reactivity present in monocytes recovered from a mouse given one AgNO₃ injection 48 hrs prior to isolation (B) or in monocytes isolated from untreated mice (C). The used rabbit antiserum recognizes both protein AA and SAA and was visualized by goat anti rabbit Alexa488-cojugated IgG. Cell nuclei were labeled with TO-PRO3. Bar 10 um.

Figure 4. SAA 1, SAA 2 and SAA 3 mRNA expression in peripheral blood monocytes were analysed with PCR.

Cells were isolated from mice that developed AA-amyloid after AEF and AgNO₃ injections or from mice that received AEF or AgNO₃ injections only or from untreated mice. Expression of the amyloid-prone SAA 1 or non-amyloidogenic SAA 2 was absent in all monocyte preparations. SAA 3 mRNA was detected in all cells independent of treatment. Mouse liver cDNA was used as a positive control. The PCR products were separated on a 1.6% agarose gel.