Background/Purpose:  
Marginal zone macrophages (MZMs) of the spleen are essential for rapid and
tolerogenic clearance of apoptotic cells (ACs).  MZMs are decreased in two
mouse models of lupus and in SLE subject spleens.  Absence of lymphotoxin beta
receptor (LTβR) signaling in the MZMs in lupus BXD2 mice leads to a defect
in megakaryoblastic leukemia 1 (MKL1)/actin polymerization.  The purpose of this
study was to determine the mechanism underlying the development of inflammatory
AC phagocytic MZMs. 

Methods:   Stimulation of LTβR
signaling was carried out in vitro with an agonistic anti-LTβR and blocked
in vivo with a soluble LTβR-Fc.  MZMs were analyzed and sorted by flow
cytometry.  Gene expression analysis was carried out by RT-PCR.  Time-lapse
imaging was carried out on a Nikon A1R two-photon microscope.  ACs were induced
by dexamethasone and labeled with DAPI.  Actin polymerization was determined
using a GFP LifeAct adenovirus transfection of macrophages (Mɸs).

Results:   Two-photon live
cell imaging revealed defects in actin polymerization and slower cytoplasmic
encapsulation of ACs at the surface of Mɸ from BXD2 and B6-Mkl1^‒⁄‒
mice.  Ingestion of ACs occurred after prolonged membrane interaction (>5
min) with ACs.  In contrast, stimulation of BXD2 Mɸ with an agonistic
anti-LTβR resulted in strong actin polymerization.  These Mɸs exhibited
robust proactive degradation of ACs characterized by cytoplasm/actin
protrusions, phagocytic cup formation, and digestion of ACs. In vivo LTβR
signaling blockade of MZMs after administration of LTβR-Fc resulted in an
increase in Il6 and a decrease in Il10.  There was also a 4-6
fold increase in the expression of serine proteases, normally found in azurophilic
granules, including neutrophil elastase (Elane), Protenase-3, and Cathepsin
G, in MZMs of BXD2 compared to B6 mice.  Increases in ELANE activity were
observed in BXD2 MZMs following AC phagocytosis or after addition of RNPs but
were suppressed by anti-LTβR. 
Gene analysis shows that there was down-regulation of the intracellular
inhibitor of TLR signaling, TRIM30α, in MZMs of BXD2, B6-Mkl1^‒⁄‒
or LTβ^f/fxCD19.Cre
mice, compared to that in B6 MZMs. Down-regulation of TRIM30α in MZMs of BXD2
and B6-Mkl1^‒⁄‒
was further verified by confocal imaging analysis.

Conclusion:   The results
show that decreased LTβR signaling in MZMs results in enhanced immunogenic
response to ACs via: (1) a defect in actin polymerization and defective AC
phagolysosomes formation; (2) a decreased repression of serine proteases that
can bind to DNA or RNA protein complexes in the endolysosome to promote
inflammatory degradation of ACs that can stimulate TLRs; and (3) a decrease in
TRIM30α which is a key inhibitor of downstream TLR signaling from
endolysosomes and Mɸ.  Since MZMs are essential for tolerogenic uptake of
ACs, the work here pinpoints specific cellular mechanistic and molecular
defects and suggest novel interventions that may reverse the immunogenic uptake
of ACs.