Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT00364728 |
| Other study ID # |
9561701025 |
| Secondary ID |
NSC98-2320-B-002 |
| Status |
Recruiting |
| Phase |
N/A
|
| First received |
August 15, 2006 |
| Last updated |
May 20, 2010 |
| Start date |
January 2006 |
| Est. completion date |
July 2012 |
Study information
| Verified date |
May 2010 |
| Source |
National Taiwan University Hospital |
| Contact |
Chung-Yi Hu, PhD |
| Phone |
886-2-2312-3456 |
| Email |
jcyhu[@]ntu.edu.tw |
| Is FDA regulated |
No |
| Health authority |
Taiwan: Department of Health |
| Study type |
Observational
|
Clinical Trial Summary
Over the past few years, growing evidences revealed that clearance of apoptotic cells by
phagocytosis can result in powerful anti-inflammatory and immunosuppressive effects. In
vivo, apoptotic cells are cleared rapidly by neighboring cells, macrophages and related
scavengers. Defective clearance of apoptotic cells has been linked closely to autoimmunity
and persistent inflammatory disease. Several phagocytic receptors, bridging molecules
produced by phagocytes and 'eat-me' signals on apoptotic cells are coordinately involved in
mediating clearance of apoptotic cells. Complement receptors (CR3, CR4), collection, CD14,
CD36 (Class B scavenger receptor), class A scavenger receptor, asialoprotein receptor, Mer
receptor kinase were reported to recognize apoptotic cells. The best characterized system
for clearance of apoptotic cells is the recognition of phosphatidylserine (PS) on apoptotic
cells by phosphatidylserine receptor (PSR). Milk fat globule- epidermal growth factor 8
(MFG-E8) is an opsonin that bridges phagocytes (by interacting with α vβ3, αvβ5 integrins
via RGD motif) and apoptotic cells (by binding PS through Factor V/VIII-C domain). Activated
macrophages produce and secret MFG-E8. MFG-E8 is a critical component in PSR-mediated
phagocytosis of apoptotic cells. The dominant negative mutant MFG-E8, D89E, that carried a
mutated RGD motif inhibited phagocytosis of apoptotic cells in vitro. Injection of D89E into
wild type mice induced autoantibodies and IgG deposition on glomeruli. Macrophages from
MFG-E8 deficiency (MFG-E8-/-) mice were impaired in engulfment of apoptotic cells, which can
be restored by adding recombinant MFG-E8. The female MFG-E8-/- mice spontaneously produced
high titer of autoantibodies and developed lupus-like glomerulonephritis at the age of week
40. Defective clearance of apoptotic cells is closely related to development of
autoimmunity. In the past 4 years, a growing number of molecules were recognized as
receptors for the PS exposed on the apoptotic cells. These molecules were capable of
mediating phagocytic clearance, rendering anti-inflammatory cytokines in the phagocytes, and
modulating T cell responses.
The specific aim of this proposal is to study genetic polymorphism in MFG-E8, PSR and other
factors implicated in phagocytic clearance of apoptotic cells among Taiwanese. By comparing
the polymorphism between patients with autoimmune disease (SLE or RA) and healthy control
subjects, we will investigate if genetic variations among individuals of genes encoding
proteins involved in clearance of apoptotic cells contribute to the pathogenesis of systemic
autoimmune diseases SLE and RA.
Description:
Over the past few years, there were growing evidences that clearance of apoptotic cells by
phagocytosis can result in powerful anti-inflammatory and immunosuppressive effects. The
defective clearance of apoptotic cells has been linked to autoimmunity and persistent
inflammatory diseases. Apoptotic cells are cleared in vivo rapidly. Clearance of apoptotic
cells or apoptotic bodies is histologically undetectable in normal situation by neighboring
cells, or by macrophages and related scavengers. Defective clearance of apoptotic cells has
been linked closely to autoimmune and persistent inflammatory diseases.
The process of discriminating apoptotic from live cells was found to be remarked complex.
Many phagocytic receptors, bridging molecules, and several 'eat-me' markers on apoptotic
cells are involved and coordinated interact with each other. A phagocytic-synapse interprets
the apoptotic-cell-associated molecular pattern (ACAMPS), and determine the behaviors
subsequent to phagocytosis.
Clearance of apoptotic cells can be mediated by complement receptors CR3 (CD116/CD18), CR4
(CD11c/CD18), collections and CD14. However, the clearance of apoptotic cells does not
usually trigger either inflammation or an adaptive immune response. Clearance of apoptotic
cells can also be mediated by receptors that were firstly characterized to clear damaged
cells or altered-self-components.
The best characterized system for clearing the apoptotic cells is the recognition of
phosphatidylerine on the apoptotic cells. Phosphatidylserine (PS) was found to distribute in
the inner layer of plasma membrane phospholipid bi-layer in healthy cells. During apoptotic
process, inhibition of aminophopholipid translocase and activation of lipid scramblase
result in exposure of PS on the cell surface.
Defective macrophage clearance of apoptotic cells linked to autoimmunity. There were
emerging evidences indicating defective macrophage clearance of apoptotic cells linked to
autoimmunity.
1. Systemic exposure to irradiated apoptotic cells induces autoantibody production. Normal
mice injected with 10^7 syngenic, apoptotic thymocytes developed transient and low
titer of anti-nuclear antibody, anti-cardiolipin, and anti-ssDNA antibody. All
immunized mice had IgG deposition in the glomeruli several months after immunization.
IgG-deposition was not found in the glomeruli of non-immunized or syngenic
splenocyte-immunized controls.
2. Defective clearance of apoptotic cells and SLE-like autoimmunity was found to be
associated C1q deficiency. C1q mice developed systemic autoimmunity spontaneously, with
a marked excess of free, non-ingested apoptotic cells of the kidney. Although
congenital deficiency of complement cascade is rare in humans, nearly all patients who
were deficient in C1q developed SLE-like disease. In both human and mice, inflammatory
macrophages had defect in the uptake of apoptotic cells and the defect could be rescued
by adding exogenous C1q.
3. Mer/kd (Mer knock-down) mice developed autoimmune disease Mer is a member of
Ax1/Mer/Tyro3 receptor tyrosine kinase family. Growth arrest-specific gene 6 (GAS6) was
shown to bridge Mer to phosphatidylserine on apoptotic cells. Mer-kd mice had
macrophages deficient in clearance of apoptotic thymocytes. The phagocytic deficiency
was restricted to apoptotic cells and was independent of Fc receptor-mediated
phagocytosis or ingestion of other particles. Mer-kd mice developed progressive
lupus-like immunity, with antibodies to chromatin, DNA, and IgG. The autoimmunity
appears to be spontaneously, driven by endogenous antigens, with little polyclonal B
cell activation.
4. Impaired phagocytosis of apoptotic material by monocyte-derived macrophages from
patients with SLE. Reduced clearance of apoptotic cells in SLE patients was observed,
although apoptosis was normally happened.
Milk fat globule-EGF factor 8 (MFG-E8) was characterized in mouse milk glycoproteins, 53 and
66 kD, peripherally associated with the membrane surrounding the lipid droplet (milk fat
globule membrane, MFGM). MFG-E8 is expressed abundantly in lactating mammary glands and
secreted in associated with milk fat globules.
MFG-E8 consists of two repeated EGF-like domains on the terminal side and tandem
discoidin-like (C-) domains homologous to the C1 and C2 domains of coagulation factor V and
VIII. The C2 domain can mediate Ca2+-independent binding to PS. MFG-E8 expression is
up-regulated in lactating mammary gland, and has been detected in other tissues such as
brain, lung, heart, kidney and spleen in mouse, bovine and human.
The second EGF-like domain of MFG-E8 contains an integrin-binding motif, namely Arg-Gly-ASP
(RGD) sequence. The RGD motif is conserved in all known MFG-E8 sequence in different
species. It can bind to αvβ3 and αvβ5 integrins. MFG-E8 was characterized as a peripheral
membrane protein, although there was no apparent hydrophobic transmembrane domain, and bind
directly to MFGM or cell membrane. The MFG-E8 binds to anionic phospholipids, especially
phosphatidylserine, on the second C (C2) domain independent of Calcium ions.
Reports indicated that MFG-E8 secreted extracellularly despite its membrane associated
nature. MFG-E8 was found to be a major component of secretory membrane vesicles (exosomes)
secreted by murine dendric cell line, D1. The glioma cell line (C6) secrets MFG-E8 into the
culture media. MFG-E8 is also detected in embryonic gonad extracellularly and in the sera of
patients with metastatic tumor.
MFG-E8 was regards as an opsonin that bridge apoptotic cells, which with surface
phoshatidylserine exposure, and phagocyte bearing αvβ3 and αvβ5 integrins. Hanayana et. al.
showed that thioglycollate-elicited macrophages produced and secreted MFG-E8. MFG-E8
significantly bound to apoptotic cells by recognizing aminophospholipids such as
phosphatidylserine and bound to macrophages via its RGD motif, particularly strongly to αvβ3
integrin. Transfected NIH3T3 cell with high αvβ5 expression can engulf apoptotic cell in the
presence of MFG-E8. The MFG-E8 dominant negative mutant that carries a point mutation in the
RGD motif inhibited phagocytosis of apoptotic cells by peritoneal macrophages in vivo and in
vitro. Borisenko et. al. estimated that MFG-E8 bind 2~8 fold stronger to oxidized PS than to
native PS. And they also proposed MFG-E8 might bind to another cofactor, annexin I, on
apoptotic cells, thus increase protein-membrane interaction.
Human MFG-E8 gene was located on chromosome 15q25. The cDNA clone contains coding sequence
for 387 ammo-acid peptides of which 263 (68%) are identical with mouse protein.
Tingible body macrophages, characterized by CD68+ F4/80-, in spleen and lymph node express
significant level of MFG-E8. Peritoneal macrophages elicited by thioglycollate secrete
abundant MFG-E8 protein of 74 kD in the culture supernatant. The macrophages from
MFG-E8(-/-) mice engulf few apoptotic cells compared to the macrophages from their normal
littermates in vitro. Addition of recombinant MFG-E8 in the culture restored the ability of
MFG-E8(-/-) Mφs to engulf apoptotic cells in a doze-dependent manner.
In germinal centers, somatic hypermutation and secondary BCR rearrangement are involved in
BCR affinity maturation. The modified BCRs with lower affinity will be removed by apoptosis.
Tingible body macrophages are responsible for the MFG-E8-dependent clearance of apoptotic
cells. Tingible body Mφs in wild type mice engulfed and digested the apoptotic B cells
efficiently, whereas the MFG-E8(-/-) Mφs just wrapped many apoptotic cells without
engulfment42. The MFG-E8(-/-) mice developed splenomegaly in an age-dependent manner. The
white pulps in the spleens of the MFG-E8(-/-) mice were greatly enlarged and carried
numerous germinal centers.
The follicular zones were enlarged. There were 2 to 3 times more lymphocytes in the spleens
of the MFG-E8(-/-) mice. An increased number of IgG-producing cells were found in the spleen
follicles. The female MFG-E8(-/-) mice spontaneously produced high titer of anti-antibodies
anti-dsDNA and ANA at the age of week 40 but not at w10. Immunization female 10-week-old
mice twice with KLH promoted ANA production 20 days later in MFG-E8(-/-) but not wild type
mice. Immune complex deposition, hyper-cellularity of glomeruli, and proteinuria were
observed in most female MFG-E8(-/-) mice at week 40. Therefore, inefficient engulfment of
apoptotic B cells by might lead to lupus-like autoimmune disease. Kenichi et al. showed that
masking phosphatidylserine by MFG-E8 mutant D89E, carrying a point mutation in RGD motif,
inhibited not only the phagocytosis of apoptotic cells by macrophages of different origins
but also the production of IL10 by thioglycollate-elicited peritoneal macrophages after
phagocytosing apoptotic cells. When D89E MFG-E8 was injected into wild type mice
intravenously, auto-antibodies were induced. The production of auto-antibodies was enhanced
by co-injection of syngenic apoptotic cells. The auto-antibodies persisted for a long-term
and IgG deposition on glomeruli took place. These results added in proof that the impairment
of phagocytic clearance of apoptotic cells leads to auto-antibody production and autoimmune
disease.In the past 4 years, a growing number of molecules were recognized as receptors for
the PS exposed on the apoptotic cells. These molecules were capable of mediating phagocytic
clearance, rendering anti-inflammatory cytokines in the phagocytes, and modulating T cell
responses.
The specific aim of this proposal is to study genetic polymorphism in MFG-E8, PSR and other
factors implicated in phagocytic clearance of apoptotic cells among Taiwanese. By comparing
the polymorphism between patients with autoimmune disease (SLE or RA) and healthy control
subjects, we will investigate if genetic variations among individuals of genes encoding
proteins involved in clearance of apoptotic cells contribute to the pathogenesis of systemic
autoimmune diseases SLE and RA.
Specific aim:
As described in the above introduction, in order to test if factors involved in clearance of
apoptotic cells are implicated in pathogenesis of human autoimmune diseases, we intend to
investigate if the genetic variation among individuals of genes encoding proteins involved
in clearance of apoptotic cells, MFG-E8 and PSR, contributes to pathogenesis of human
autoimmune diseases SLE and RA.
The goal of this proposal:
We will
1. Find out genetic polymorphism of human PSR gene in Taiwan by study single nucleotide
polymorphism (SNPs) of PSR genes among Taiwanese subjects using PCR and DNA sequencing.
2. Study the PSR SNPs in SLE and RA patients and non-auto-immune control subjects by
PCR/sequence-specific oligonucleotide probe hybridization.
3. Compare the allelic distribution of each SNP among patients and control group and find
if there is disease-associated SNP(s) in PSR gene.
4. Find out genetic polymorphism of human MFG-E8 gene in Taiwan by study SNPs of MFG-E8
genes among Taiwanese subjects using PCR and DNA sequencing.
5. Study the MFG-E8 SNPs in SLE and RA patients and non-auto-immune control subjects by
PCR/sequence-specific oligonucleotide probe hybridization.
6. Compare the allelic distribution of each SNP among patients and control group and find
if there is disease-associated SNP(s) in MFG-E8 gene.
