Yves Lombard

Uptake of Aβ 1–40- and Aβ 1–42-coated yeast by microglial cells: a role for LRP

Artificial diffuse and amyloid core of neuritic plaques [β‐amyloid peptide (Aβ) deposits] could be prepared using heat‐killed yeast particles opsonized with Aβ 1–40 or Aβ 1–42 peptides. Interaction and fate of these artificial deposits with microglial cells could be followed using a method of staining that allows discrimination of adherent and internalized, heat‐killed yeast particles. Using this system, it was possible to show that nonfibrillar or fibrillar (f)Aβ peptides, formed in solution upon heating (aggregates), could not impair the internalization of heat‐killed yeast particles opsonized with fAβ 1–40 or fAβ 1–42. This indicated that depending on their physical state, Aβ peptide(s) do not recognize the same receptors and probably do not follow the same internalization pathway. Using competitive ligands of class A scavenger receptors (SR‐A) or low‐density lipoprotein‐related receptor protein (LRP), it has been shown that SR‐A were not involved in the recognition of amyloid peptide deposits, whereas LRP specifically recognized deposits of fAβ 1–42 (but not fAβ 1–40) and mediated their phagocytosis.

Amyloid β Peptides Trigger CD47-Dependent Mast Cell Secretory and Phagocytic Responses

Mast cells are found in the brain, where they contribute to immune responses. They have been implicated in multiple sclerosis, but their potential role in Alzheimers disease (AD), another inflammatory disease of the central nervous system, remains elusive. In the present study, we examined mast cell responses to amyloid β (Aβ) peptides 1-40 and 1-42, the major components of the Alzheimer amyloid plaques. Rat peritoneal mast cells were used as experimental model for human brain serosal mast cells. Fibrillar Aβ1-40 and Aβ1-42 peptides induced concentration-dependent exocytosis, as assessed by measurement of histamine secretion; exocytosis was reduced by pre-treatment with pertussis toxin and with antibodies against the CD47 receptor and the β1-integrin subunit. Fibrillar Aβ1-40 and Aβ1-42 peptides coated on heat-inactivated yeast particles and soluble fibrillar Aβ1-40 and Aβ1-42 peptides were also recognized and phagocyted by mast cells. Uptake of the peptides was decreased in the presence of 4N1, a peptide agonist of the CD47 receptor, but remained unchanged in the presence of 4NGG, a peptide derived from 4N1 which does not bind to CD47. Non-fibrillar forms of Aβ1-40 and 1-42 peptides were unable to elicit mast cell responses. These results show that fibrillar Aβ peptides can trigger mast cells and elicit exocytosis and phagocytosis. The Aβ-induced activation of mast cells operates through a CD47/β1-integrm membrane complex coupled with Gi.-protein. The present data support the hypothesis that mast cells, similarly to microglial cells, could play a major role in AD pathogenesis.

Complement receptor 3 (CD11b/CD18) is implicated in the elimination of β-amyloid peptides.

Microglia are the professional phagocytes of the brain and express phagocytic receptors such as complement receptor 3 (CR3 or CD11b/CD18). Using mimics of the amyloid deposit made of heat‐killed yeasts coated with either Aβ 1‐40 or Aβ 1‐42, we were able to study how microglia interacted with and ingested these particles in vitro. We have shown previously that the low density lipoprotein receptor‐related protein (LRP) is largely implied in the phagocytosis of Aβ 1‐42‐opsonized heat‐killed yeasts and partly in that of Aβ 1‐40‐opsonized heat‐killed yeasts. Here, we report that antibodies against CD11b or CD18 reduced the uptake of the artificial amyloid deposit by microglial cell showing that CR3 is involved in the mechanism. Moreover, a concomitant inhibition of LRP and CR3 completely blocked the ingestion of both kinds of particles suggesting that no other receptors participate to this mechanism.

The role of calcium and magnesium ions in uptake of beta-amyloid peptides by microglial cells.

Amyloid peptides 1-40 and 1-42 (Aβ 1-40 and Aβ 1-42) are major components of diffuse and neuritic senile plaques present in the brain of patients with Alzheimers disease. Their interaction with microglial cells was studied using a system partly mimicking these plaques, which consisted in heat-killed yeast particles coated with either Aβ 1-40 or Aβ 1-42. Using these particles, it has been shown in our laboratory that LRP is involved mainly in the elimination of Aβ 1-42-coated heat-killed yeast particles and partly in that of Aβ 1-40-coated heat-killed yeast particles by microglial cells in culture. We show here that in the presence of calcium and magnesium ions extracellular chelators, namely EDTA (for both ions) and EGTA (for calcium ions), the internalization of coated heat-killed particles was impaired. In the presence of BAPTA-AM, an intracellular chelator of calcium ions and thapsigargin, an inhibitor of the endoplasmic reticulum calcium pump, no effect was observed on the phagocytosis of Aβ 1-40-coated heat-killed yeast particles, whereas that of Aβ 1-42-coated heat-killed yeast particles was affected. These results suggest that different signaling mechanisms are involved after the internalization of Aβ 1-40 and Aβ 1-42.

Characterization of surface markers of continuously growing murine resident macrophages.

Conditions have been described which allow an in vitro indefinite multiplication of differentiated murine macrophages (Lombard et al: Biol Cell 53, 219, 1985). R. and MAY‐1 ceil lines, which were obtained, respectively, from mouse (Balb/c) spleen and resident peritoneal macrophages, have been further characterized. They present at their surface, besides the Mac‐1 antigen and Fc‐receptor, a mannose receptor which was characterized for its binding properties. This receptor Is responsive for a specific phagocytosis of mannosylated particles, i.e., mannosylated latex beads or oil droplets containing mannosylated bovine serum albumin. Moreover, R and MAY‐1 ceils present an ectoenzyme profile (NAD+ glycohydrolase and nucleotide pyrophosphatase) similar to those of the corresponding resident macrophages.

Phagocytic functions of microglial cells in the central nervous system and their importance in two neurodegenerative diseases: multiple sclerosis and Alzheimer’s disease

Microglial cells are the resident phagocytic cells of the central nervous system (CNS). They possess a wide range of receptors allowing them to identify and internalize numerous pathogens. We will discuss here the role of the most important receptors of microglia involved in non-opsonin-dependent phagocytosis (mannose receptor, β-glucan receptor, scavenger receptor) and that of receptors involved in the opsonin-dependent phagocytosis, namely the complement 3 (CR3) and the Fcγ receptors (FcγR). First, the molecular and cellular mechanisms induced when these receptors are conducting a phagocytic event are presented. In the second part, we will discuss the role these receptors may play in multiple sclerosis and Alzheimer’s disease, in the elimination by phagocytosis of myelin and beta amyloid peptide respectively.

Cell Culture Models for Mononuclear Phagocytes

Mononuclear phagocytes are ubiquitous cells and practically all tissues can be used as sources for obtaining them, most commonly: bone marrow, thymus, spleen, blood, peritoneal cavity, lungs, brain, skin. In this chapter, particular attention will be focused on the primary culture models developed from murine peritoneal macrophages and human peripheral blood monocytes; the use of these systems is recommended by the Society for Leukocyte Biology (Morahan 1980). The description of methods for obtaining other types of mononuclear phagocytes or f studying them can be found in specialized handbooks (Adams 1979 Adams et al. 1981; Beelen and Poindron 1994).