Macrophages play important roles not only in the host defense
but also in allergic reactions. In many types of immunologic
inflammatory lesions including synovial tissue of rheumatoid arthritis
(RA), (
)macrophages are the predominant cells in the
leukocyte infiltrate(1) ; however, in certain situations such
as the reaction in synovial cavities of RA, macrophages are only a
minor population, and polymorphonuclear leukocytes (PMNs) are most
predominant(2, 3) . The majority of the macrophages in
the extravascular foci of inflammation are thought to be derived from
blood monocytes, being attracted by chemotactic factors produced
locally. A number of molecules possessing the chemoattracting capacity
to monocytes are now known; for instance, complement C5a(4) , a
complement factor H derivative(5, 6) , monocyte
chemoattractant protein-1 (MCP-1) (7) ,
tetranectin(8) , and formyl-methionyl
peptides(9, 10) . However, many of them attract PMNs
as well. To explain the different histopathological features regarding
the macrophage infiltration in synovial tissues versus synovial cavities of RA as described above, we have been
investigating chemical mediators that participate in the phenomena. We
have already reported the presence of a strong inhibitory factor to the
monocyte chemotaxis in RA-synovial fluid(11, 12) ,
which seemed to be responsible for the PMN-predominant/monocyte-poor
infiltration in RA-synovial cavities.
Additionally, we have
described the presence of a monocyte chemotactic factor in human serum (13) as well as in guinea pig serum(14) , which is
generated concurrently with plasma clotting. This factor does not
attract PMNs. The monocyte-specific chemotactic factor is initially
present as a precursor molecule. It is converted to the active form by
a transglutaminase-catalyzed reaction(13) .
The initial
purpose of the present project was to analyze and identify a monocyte
chemotactic factor(s) that would play a major role in the monocyte
predominant infiltration in the RA-synovial tissue. In this report, we
describe a surprising result that the major monocyte specific
chemotactic factor in RA-synovial tissue seems to be S19 ribosomal
protein (RP S19) itself or its homologue cross-linked by the
transglutaminase catalyzed reaction.
EXPERIMENTAL PROCEDURES
Materials and Reference Compounds
Hank's
balanced salt solution (HBSS) and RPMI 1640 medium were purchased from
Nissui Pharmaceutical Co. (Tokyo, Japan). Bovine serum albumin,
cytochrome-C, ovalbumin, bovine
-globulin, zymozan, dns-cadaverine
(lysine doner) and p-chloromercuriphenyl sulfonic acid were
purchased from Sigma. Purified human factor XIII was a gift from
Institute of the Chemo Sero Therapy (Kumamoto, Japan). Toyopearl HW-55S
and CM-Toyopearl 650 M were obtained from Tosoh Company
(Tokyo, Japan). DEAE-Sephadex A-50, protein G-Sepharose 4 Fast Flow, a
Superdex 200 HR 10/30 column, CNBr-activated Sepharose 4B beads, and
ampholine were purchased from Pharmacia Biotech Inc. Mouse anti-human
MCP-1 monoclonal antibody was a gift from Dr. Yoshimura (National
Cancer Institute). Anti-human complement C5 sheep IgG antibody was a
product of Birmingham Research Institute (Birmingham, UK). A multiwell
chamber for chemotaxis assay was obtained from Neuro Probe (Bethesda,
MD). Nuclepore filters were purchased from Nuclepore (Pleasanton, CA).
Elastatinal, leupeptin, E-64, phosphoramidon, diisopropyl
fluorophosphate, pepstatin, and N-ethylmaleimide were
purchased from Peptide Institute (Osaka, Japan). D-Phe-Pro-Arg-chloromethyl ketone was purchased from
Calbiochem (La Jolla, CA). Sodium deoxycholate was purchased from
Difco. 6-Amino-n-caproic acid and all other chemicals were
obtained from Wako Pure Chemicals (Osaka, Japan). Zymosan-activated
human plasma (ZAP) was prepared according to the method of Fernandez (4) with a modification as described previously(11) .
IgG antibodies against human complement C4 or C5 and cytokine MCP-1
were coupled to CNBr-activated Sepharose 4B beads, respectively, by the
method of Porach and Axen(15) .
Preparation of RA-synovial Tissue
Extract
RA-synovial tissues were surgically removed from the
knee joint of RA patients. They were immediately frozen and weighed (g)
and then kept at -80 °C until use. They were sliced at 50
µm thick using a cryomicrotome and extracted for 4 h at 4 °C
with phosphate-buffered saline (PBS), pH 7.2, containing 0.1 mg/ml
polybrene, 10 mM EDTA, 5 mM
6-amino-n-caproic acid, 1 mMp-amidinophenylmethanesulfonyl fluoride hydrochloride (p-APMSF), 0.1 mM elastatinal, 0.1 mM chymostatin, 0.1 mM leupeptin, 0.1 mM E-64, 0.1
mM phosphoramidon, and 0.1 mM bestatin at a ratio of
250 ml of the PBS solution to 100 g of tissue. After centrifugation at
13,000 rpm for 20 min at 4 °C, the supernatant was used as
RA-synovial tissue extract.
Assay of Transglutaminase Activity
The
transglutaminase activity of factor XIIIa was measured by a modified
method of the Lorand et al.(16) using an assay kit,
Iatro-FL F XIII
(Iatron Laboratories, Tokyo, Japan). One
unit was defined as the activity contained in one ml of normal human
plasma as factor XIII.
Chemotaxis Assays
Monocytes and PMNs were isolated
from heparinized human venous blood of healthy donors according to the
method of Fernandez et al.(4) as described
previously(11, 12) . The monocytes and PMNs were
suspended at a concentration of 1
10
cells/ml in
HBSS containing 0.5% bovine serum albumin or in RPMI 1640, pH 7.2, for
the morphologic polarization assay or for the multiwell chamber assay.
The morphologic polarization assay was performed according to the
method of Cianciolo and Snyderman (17) as described
previously(11, 12) . Unless otherwise specified, the
assay samples were diluted at least 10-fold with HBSS prior to the
assay. As a positive control and a negative one, 1% (v/v) ZAP, and HBSS
were used as chemoattractants, respectively. The activity of the
samples was initially calculated as the percentage of monocytes or PMNs
with polarized morphology composed with the total numbers of cells
counted. The ratio of polarized cells in percentage was proportional to
the logarithmic change of concentration of RA-synovial tissue extract
or ZAP when the concentrations were low (below 10%). Polarization
activity was converted and expressed by an arbitrary unit; one
polarization unit corresponded to the activity contained in 1% ZAP (12) . The multiwell chamber was used according to the method
of Falk et al.(18) , using a Nuclepore filter with a
pore size of 5 µm. After the incubation, the membrane was
separated, fixed with methanol, and stained with Giemsa. The total
number of monocytes that had migrated beyond the lower membrane was
counted in five high power fields. As a positive control and a negative
one, 1% (v/v) ZAP, and PBS were used as the chemoattractants,
respectively. Results were expressed as the number of migrated
monocytes. Checkerboard analysis of the monocyte chemotactic factor was
carried out by the method of Zigmond and Hirsch (19) utilizing
the multiwell chamber assay.
Isoelectric Focusing
A sample that was diluted
with distilled water containing 1% ampholine (a mixture of pH
3.5-9.5, pH 7-9, and pH 9-11 at an equal volume) to
the final volume of 30 ml were applied to the Rotofor(TM) cell
(Bio-Rad). After focusing under a constant electric power at 12 watts
for 8 h at 4 °C, the solution was fractionated into a volume of 1.5
ml. In each fraction, pH and the monocyte polarization activity were
measured.
NH
-terminal Amino Acid Sequence
Analysis
Limited NH
-terminal amino acid sequencing
was carried out in duplicate for the initial 20 cycles with a protein
sequencer (Applied Biosystems, 477A), equipped with a
phenylthiohydantoin-derivative analyzer (Applied Biosystems, 120A),
according to the instrument manual. The homology search for the amino
acid sequence was carried out using DBGET integrated data base
retrieval system, Swiss-Prot.
SDS-Polyacrylamide Gel Electrophoresis
(PAGE)
Electrophoresis was carried out on a vertical slab gel of
15% polyacrylamide according to the method of Laemmli(20) . The
samples were boiled for 2 min and applied to the gel. After
electrophoresis at 25 mA for 40 min, the gel was stained by either the
silver or Coomassie Blue staining method.
Preparation of Ribosomal Protein
Fractions
Preparation of ribosomes from bovine liver was carried
out by the method described previously(21) . The ribosomal
fraction was then separated to 40 and 60 S subunits as described
previously(22, 23) . Ribosomal proteins of the 40 and
60 S subunits were isolated from RNAs with 66% acetic acid in the
presence of 0.1 M MgCl
as described
previously(24, 25) . We obtained 10 mg of protein (at
a concentration of 100 µg/ml) in each subunit fraction from 500 g
of bovine liver. After dialyzation against PBS, the ribosomal protein
fractions were used in the present experiments.
Preparation of Recombinant RP S19-Maltose Binding Protein
Fusion Protein
cDNA was prepared from HepG2 cell mRNA by the
common method using reverse transcriptase oligo(dT) and randam primer
(Takara). Two primers consisting of nucleotides based on cDNA sequences
of human RP S19 were obtained from EMBL sequence data base; (i) a sense
primer consisting of nucleotides 1-30, namely
5`-ATGCCTGGAGTTACTGTAAA-3` and (ii) an antisense primer consisting of
nucleotides 423-453 in which G
and T
were changed to C and A, respectively to construct the HindIII restriction site, namely
5`-GTTGTTCTTCGTAATCTTGTTTCGAACGAC-3`. Using these primers and HepG2
cDNA, nucleotides coding the full length of the human RP S19 were
prepared by the polymerase chain reaction with Puf DNA
polymerase (Stratagene)(26, 27) . The RP S19
nucleotides were processed with HindIII and were ligated to pMAL-c2 vector, which had been processed with XmnI
and HindIII. The constructs were transfected into the
competent Escherichia coli, JM109 cells by the heat shock
method, and positive transformants were cloned. One of the positive
clones was cultured, and the correct insertion of RP S19 was confirmed
by making the restriction map. The cloned JM109 E. coli cells
were cultured in Rich medium containing glucose (2 g/liter) and
ampicillin (100 mg/liter) and stimulated to synthesize the recombinant
protein by adding isopropyl-1-thio-
-D-galactopyranoside
for 2 h at 37 °C. After washing by centrifugation for 20 min at
2,500 rpm, the JM109 cells were resuspended in a buffer (10 mM Tris-HCl containing 200 mM NaCl, 1 mM EDTA, 1
mM sodium azide, and 1 mM dithiothreitol), which
could then be used for amylose resin column chromatography. The JM109
cells were broken by sonication, and periplasmic products were
separated by centrifugation at 6,400 rpm for 30 min into the
supernatant. This supernatant was applied to the amylose resin column.
After extensive washing of the column, the recombinant fusion protein,
MBP-RP S19 was eluted with 10 mM maltose in the same buffer.
MBP-RP S19 was further purified by the Superdex 200 HR 10/30 column
equilibrated with 10 mM Tris-HCl containing 20 mM NaCl, pH 7.5. The major fraction with an apparent molecular size
of 60 kDa was used in the following studies.
RESULTS
Monocyte-directed Migration Activity of RA-synovial
Tissue Extract
We examined migration attracting potencies of the
RA-synovial tissue extract to monocytes and PMNs using the morphologic
polarization assay. RA-synovial tissues obtained from seven patients
were individually extracted, and the seven RA-synovial extracts were
assayed separately. The results are shown in Fig. 1as the mean
values. The RA-synovial extracts possessed the polarization activity
for monocytes but not for PMNs. Since the differences among the samples
were very small in regard to the polarization activities, a mixture of
the extracts obtained from the seven RA-patients was used in the
following experiments.
Figure 1:
Polarization
inducing activity of RA-synovial tissue extract to monocytes and PMNs.
Monocytes or PMNs were attracted with various concentrations of seven
individual RA-synovial tissue extracts. The concentration on the horizontal axis means the final concentration of the samples.
The lines with the closed circles or with the open circles denote the polarization responses of monocytes or
PMNs, respectively. Values are mean ± S.D. (Three experiments
were carried out for each extract).
Brief Analysis of Monocyte Migration Factors in
RA-synovial Tissue Extract
Molecular size analysis of monocyte
migration factors in the RA-synovial tissue extract by gel permeation
HPLC was carried out using the Toyopearl HW-55S column. As shown in Fig. 2a, two peaks of monocyte polarization activity were
observed. One peak with larger molecular mass was much stronger than
the other peak with smaller molecular mass. The major peak fraction did
not induce the polarized shape change of PMNs, while the other
possessed an obvious activity to PMNs as well (data not shown). The
monocyte polarization activity in the major fraction was totally
absorbed by the anti-complement C5 antibody immunoadsorption beads,
whereas activity in the minor peak fraction was partially absorbed
either by the anti-MCP-1 antibody beads or by the anti-complement C5
antibody beads in a batch-wise method for 30 min at 20 °C (data not
shown).
Figure 2:
Nature of monocyte migration factors in
RA-synovial tissue extract. a, an aliquot (3 ml) of a pool of
the seven RA-synovial tissue extracts was applied to the column
(Ø, 20
1,000 mm; bed volume, 314 ml) equilibrated with
PBS containing 10 mM EDTA, 10 mM
6-amino-n-caproic acid, and 0.5 mMp-APMSF,
pH 7.2. The flow rate was 1 ml/min, and the eluate was fractionated at
2.5 ml. The solid line, the hatched columns, and the arrows denote the absorbance at 280 nm, the monocyte
polarization activity, and the eluted positions of the molecular size
markers, respectively (from A to D; bovine
-globulin, 150 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43
kDa; cytochrome c, 12 kDa; respectively), respectively. b, the pool (380 ml) of the breakthrough fraction in the
DEAE-Sephadex column chromatography of RA-synovial tissue extract was
dialyzed against 10 mM phosphate buffer, pH 6.0, containing 40
mM NaCl and applied to a column (Ø, 23
160 mm;
bed volume, 67 ml) equilibrated with the same buffer. The elution was
carried out by a NaCl gradient at 4 °C at a flow rate of 10
ml/cm
h. The broken line denotes the
theoretical concentrations of NaCl (mM). The double-headed arrow indicates the fractions pooled
for the next chromatographic step. c, an aliquot (240 µl)
of the breakthrough fraction of the protein G column chromatography was
applied to the column (Ø, 10
300 mm; bed volume, 24 ml)
equilibrated with PBS, pH 7.2. The flow rate was 0.5 ml/min, and the
eluate was collected in 1-ml fractions. The thick solid line denotes the absorbance at 235 nm.
Separation and Characterization of the Major Monocyte
Migration Factor in RA-synovial Tissue Extract
The RA-synovial
tissue extract (385 ml) was dialyzed against 10 mM phosphate
buffer containing 40 mM NaCl, and 1 mM EDTA (pH 7.5)
and subjected to anion-exchange column chromatography with
DEAE-Sephadex A-50 (Ø, 25
390 mm; bed volume, 190 ml)
equilibrated with the same buffer, at a flow rate of 12
ml/cm
h at 4 °C. The majority (about 80%) of the
monocyte polarization activity passed through the column (data not
shown). The active breakthrough fraction was dialyzed and applied to a
cation exchange column with CM-Toyopearl 650 M. As shown in Fig. 2b, the monocyte polarization activity totally
bound to the column and was eluted with 140 mM NaCl in the
gradient elution. The active fractions were pooled (280 ml) and applied
to a protein G-Sepharose 4 Fast Flow column equilibrated with 10
mM phosphate buffer containing 200 mM NaCl (pH 6.0)
at a flow rate of 9 ml/h at 4 °C. The whole activity passed through
the column (data not shown). An aliquot of the breakthrough fraction of
the protein G-Sepharose column chromatography was applied to the
Superdex 200 HR 10/30 gel filtration column. As shown in Fig. 2c, the monocyte polarization activity was eluted
as a single peak in a fraction, which corresponded to a molecular mass
of 45 kDa. Using the active fraction of this gel permeation HPLC, the
isoelectric point of the monocyte migration factor was examined. The
monocyte polarization activity was focused at pH 8.1 (data not shown).
Promotions of monocyte migration are classified into two categories,
chemotaxis and chemokinesis. The effect of the monocyte migration
factor was studied in this regard by the checkerboard analysis with the
multiwell chamber. As shown in Table 1, the monocyte migration
factor was only effective when the concentration of the molecule was
higher in the lower well than in the upper well, where the indicator
cells were present. These results indicate that the effect of the
monocyte migration factor was chemotaxis but not chemokinesis.
Purification of the Monocyte Chemotactic
Factor
The remainder of the breakthrough fraction in the protein
G column chromatography was applied to an anti-human complement C5
antibody column. The monocyte polarization activity was absorbed by the
column except for a minor fraction (about 5% of the activity applied).
After washing the column, the chemotactic factor was eluted with 0.1%
trifluoroacetic acid. The active fraction eluted from the anti-C5
antibody column was pooled and analyzed by a reverse phase HPLC with
the C4 column. A sharp single peak of absorbance at 235 nm associated
with the monocyte chemotactic activity was eluted at an acetonitrile
concentration of 47%. The molecules in the breakthrough possessing the
absorbance at 235 nm had neither protein nature in the spectrum
analysis nor chemotactic capacity. In the purification process, 5
µg of protein was obtained from 380 ml of the RA-synovial tissue
extract.
NH
-terminal Amino Acid Analyses
The
protein eluted from the C4 column was analyzed by the protein
sequencer. As shown in Fig. 3, the 20 NH
-terminal
amino acids of the synovial chemotactic factor were completely
overlapped with those of human RP S19.
Figure 3:
NH
-terminal amino acid
sequence of the monocyte chemotactic factor. The chemotactic factor
eluted from the C4 column was analyzed by the protein sequencer for the
initial twenty amino acid residues. Human RP S19 was picked up by the
homology search using EMBL nucleic acid sequence data base. Amino acid
sequence of the two proteins is comparatively
shown.
SDS-PAGE Analysis
As shown in Fig. 4, the
peak fraction of the reverse phase HPLC demonstrated two silver
staining bands in SDS-PAGE. The major band possessed the apparent
molecular mass of 34 kDa and did the minor one of 68 kDa. These
molecular sizes are double and quadruple of the size of RP S19 (16
kDa).
Figure 4:
SDS-PAGE of the purified monocyte
chemotactic factor. Approximately 30 ng of protein of the reverse phase
HPLC fraction was electrophoresed in a 15% polyacrylamide gel and
visualized by silver staining. The positions corresponding to three
marker proteins are shown at the left side (67 kDa, bovine serum
albumin; 43 kDa, ovalbumin; 30 kDa, carbonic anhydrase,
respectively).
Potential Monocyte Chemotactic Activity in Ribosomal
Protein Fraction
It is known that RP S19 is a component of 40 S
ribosome subunit. Ribosomal protein fractions of 40 and 60 S subunits
were prepared from bovine liver. Neither of the 40 S ribosomal protein
fraction nor the 60 S one elicited monocyte chemotaxis in the multiwell
chemotaxis assay. These ribosomal protein fractions were treated with
factor XIIIa (at a final concentration 1 unit/ml) or the same volume of
PBS, pH 7.2, in the presence of 5 mM CaCl
for 20
min at 37 °C. After this treatment, the 40 S ribosomal protein
fraction was capable of attracting monocyte chemotaxis. In contrast to
this, the 60 S fraction exhibited a neglegible activity even after the
treatment with the transglutaminase (data not shown).
Generation of Monocyte Chemotactic Activity using MBP-RP
S19 Recombinant Protein
The MBP-RP S19 was incubated with factor
XIIIa (final concentration, 1 unit/ml) for 90 min at 37 °C. As
shown in Fig. 5a, a significant amount of the monocyte
chemotactic activity was observed after the incubation, although before
the incubation, only negligible activity was observed. In SDS-PAGE
analysis (Fig. 5b), with factor XIIIa, a band with an
apparent molecular size 120 kDa newly appeared. Since the apparent
molecular size of this band is double of MBP-RP S19 (60 kDa), the new
band is thought to be a dimer of MBP-RP S19. The same experiments were
carried out using simple MBP instead of MBP-RP S19. Neither
dimerization nor chemotactic activity generation was observed (data not
shown). To confirm that the dimeric MBP-RP S19 bearing the chemotactic
activity was produced by the isopeptide bond formation between a
glutamine and lysine residues, a competitive inhibition study was
carried out with a synthetic substrate of factor XIIIa. The same
experiment was performed except for the presence of dns-cadaverine
(lysine donor) and for a shorter incubation period (20 min). As shown
in Fig. 6, the generation of the monocyte chemotactic activity
significantly decreased as the dns-cadaverine concentration increased.
Figure 5:
Factor XIIIa-mediated monocyte chemotactic
activity of RP S19. MBP-RP S19 (final concentration 1
10
M) was incubated with factor XIIIa
(final concentration, 1 unit/ml) and 5 mM CaCl
at
37 °C for 90 min. a, hatched columns denote monocyte
chemotactic activity by chemotaxis assay using 48-well chemotaxis
chamber. Vertical axis denotes the number of migrated
monocytes beyond the lower membrane. Values are mean ± S.D.
(Three experiments were carried out for each examination). b,
samples (lane 1, 0 min; lane 2, 90 min) were
electrophoresed in a 15% polyacrylamide gel, and stained by Coomassie
Blue. The positions corresponding to factor XIIIa (80 kDa) and MBP-RP
S19 (60 kDa) are shown at the right side.
Figure 6:
Factor XIIIa-mediated monocyte chemotactic
factor. MBP-RP S19 (final concentration 0.5
10
M), which was mixed with factor XIIIa (final
concentration, 1 unit/ml) and 5 mM CaCl
was
incubated with several concentration of dns-cadaverin at 37 °C for
20 min. Closed circles denote the monocyte chemotactic
activity. The horizontal axis denotes the final concentration
of dns-cadaverin (M).
DISCUSSION
Reflecting the histological picture of RA-synovial tissue,
the chemotactic potency of all of the RA-synovial tissue extracts
obtained from seven patients was much more potent for monocytes than
for PMNs. As expected from previous immunohistochemical
studies(1) , multiple molecules that possess chemotactic
capacity to monocytes were indeed present in the extract of the
RA-synovial tissues including MCP-1 and C5a or its derivative (data not
shown). However, the major chemotactic factor in the extract was not
these previously known factors. The major factor seems to be a
homodimer (to some extent a homotetramer) molecule of RP S19 itself or
its homologue for the following reasons:
1) The
NH
-terminal sequence of 20 amino acid residues of the
chemotactic factor was identical to that of S19 ribosomal protein from
the second residue to the 21st residue (Fig. 3). In our homology
search using Swiss-prot amino acid sequence data base, only RP S19 has
this sequence. 2) The recombinant MBP-RP S19 exhibited monocyte
chemotactic activity after being dimerized by the treatment with factor
XIIIa. 3) The RP fraction of bovine liver 40 S subunit, which would
contain RP S19, exhibited monocyte chemotactic activity after the
treatment with factor XIIIa. 4) The apparent molecular mass (34 and 68
kDa) of the monocyte chemotactic factor in the SDS-PAGE analysis (Fig. 4) were double and quadruple of that (16 kDa) of RP S19.
(v) RP S19 possesses many glutamine (eight) and lysine (15) residues, which could be substrate residues of
transglutaminase.
The monocyte chemotactic factor bound to the
anti-C5 antibody beads. In the amino acid sequence of RP S19, no
homologous portion to the sequence of C5 is present. Despite a lack of
evidence, our speculation is that a tridimensional structure similar to
C5a (C5-derived leukocyte chemotactic peptide) might appear on the
dimer and oligomer molecules of RP S19 as a consequence of the
transglutaminase-catalyzed intermolecular cross-linking.
We have
previously reported the presence of a precursor molecule of monocyte
chemotactic factor in plasma, which is converted to the active molecule
by the transglutaminase-catalyzed reaction during plasma
clotting(13) . The relationship between the monocyte
chemotactic factor in serum and RA-synovial tissue extract has not been
fully elucidated. However, in consideration of their molecular mass and
isoelectric point, they seem to be identical molecules. (
)A
small amount of RP S19 might be present in the circulation. It is
obvious that factor XIIIa plays a role as a transglutaminase to
generate the monocyte chemotactic factor in serum. However, we cannot
be sure whether factor XIIIa or cellular transglutaminase as the
transglutaminase in the the monocyte chemotactic factor formation in
RA-synovial lesion. Recently, production large amounts of
transglutaminase in monocyte-derived antigen-presenting cells was
reported(28) , and indeed the transglutaminase antigen is used
for immunohistochemical identification of these cells(29) .
However, the presence of the monocyte-derived antigen-presenting cells
in RA-synovial lesion has been well documented (1) . Therefore,
it is possible that the catalyst to construct the monocyte chemotactic
factor in RA-synovial tissue is at least in part the cellular
transglutaminase liberated from the blood monocyte-derived cells.
Therefore, one could speculate the presence of a positive feedback
circuit causing the monocyte/macrophage predominant infiltration via
generation of the monocyte chemotactic factor by macrophage-derived
transglutaminase in RA-synovial tissue lesions.