BIOCHEMICAL
Vol. 172, No. 3, 1990 November 15, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1203-1208
EXUDATION INDUCES CLUSTERING OF CR1 RECEPTORS AT THE SURFACE OF HUMAN POLYMORPHONUCLEAR LEUKOCYTES J.-P. Paccaud,J.-L. Carpentier, and J.A. Schifferli* Institute of Histology andEmbryology, University Medical Center, Geneva, *Division of Nephrology, Departmentof Medicine, Geneva, Switzerland Received
September
24,
1990
The complementreceptor type 1 (CRl) surfacedistribution, density and immuneadherence efficiency were determined in circulating PMN activated by fMLP, NAP-l/IL-8, TNF, GM-CSF and C5a, or exudate PMN harvestedfrom skin-blisters.Theseobservationswere comparedwith thoseobservedon restingpet-i-pheralblood PMN. PMN activators known to upregulateCR1 expressiondid not induce a significant increase in CR1 clustering, or immuneadherenceefficiency towardsopsonizedimmunecomplexes. By contrast, increasein CR1 density at the surfaceof exudatedPMN was accompaniedby an increasedclustering. This clustering was however insufficient to increasethe binding efficiency for immune complexes. Eventually, CR1 expression of exudated neutrophil could not be increasedfurther by stimulationwith fMLP or PMA. Theseresults indicated that clustering of CR1 on PMN may occur in vim. Such reaction might determine the phagocytic potential of the cell for opsonized micro-organismsor debris.This clusteringcould not be attributed to one of the PMN activators tested. 0 1990 Ac*demlc Press,Inc. Upon activation, human polymorphonuclear leukocytes (PMN) upregulate complementreceptorstype 1 (CRl, CD35) (l), which are involved in immune adherence reactions, aswell asin phagocytosisof complement-reactedparticles (2,3). However, the efficiency of immune adherencereactionsdoesnot appearto dependon the increasedCR1 density, but rather on the degreeof CR1 clustering. Indeed, in vitro activation of PMN by the chemotactic factor fMLP which increasesthe surface density of CRI, but not the proportion of clusteredreceptors(4), leadsto a poor binding efficiency ascomparedto that of erythrocytes, known to bear clusteredCR1 (5). Several substancessusceptibleto be found at inflammatory sitessuchasC5a, LTB4, PAF (1,6,7), NAP-l/IL-8
(8), or tumor necrosis factor (TNF) (9) are also known to
upregulateCRl. Furthermore, complementreceptorsare increasedon the surfaceof PMN in inflammatory exudates (10). We comparedthe surface distribution of CR1 in exudate and peripheral blood PMN and showthat migration into an exudate leadsto an increasein CR1 clustering, increasethat could not be mimicked in vitro by severalknown upregulators of CRl. METHODS Buffers and reagents. Ficoll 400 and Dextran T500 were obtained from Pharmacia.Bovine serum albumine (BSA), N-formyl-methionyl-leucyl-phenylalanine 0006-291
1203
Xi90
$1.50
Vol.
172, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
(fMLP), phorbol meristate acetate (PMA) from Sigma. Purified human C5a was kindly provided by Dr. Dahinden, Institute of Clinical Immunology, University of Bern. Human recombinant Tumor NecrosisFactor (TNF) was a gift from Dr. G. Grau, Departmentof Pathology, University Medical Center, Geneva. Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) was kindly provided by Dr. J.-J. Mermod, Glaxo, Geneva. [125-1]radiolabeled monoclonalantibody El 1 directed againstCR1 was usedto quantitate CR1 sites on PMN (4). Complement-reacted Hepatitis B surface antigen immune complexes (IC) were prepared asdescribed(4). Phosphatebuffer saline,pH 7.4 , as such (PBS), or supplementedwith 5mg/ml BSA (PBSB) wasused. Cell purification. Resting blood PMN were purified at 4’C (4). Exudate PMN were collected asfollowed: after ethanol disinfection, 4 skin blisterswere produced on the thigh by a suction chamber to which a vacuum of -200 mmHg was applied for 3 hours. The top of the blisters were removed and the lesionscovered by a sterile adhesiveplastic film (Opsite). After approximately 15 hours, the fluid under the plastic film was collected with a syringe. Between 5 to 25~10~cells were obtained with this method, 95% of which were PMN. After washingthe exudatein ice cold PBSB, viability was assessed by Trypan Blue exclusion and wasalways greater than 97%. CR1 measurement and IC binding assays were performed as previously described(4). Briefly, cells (with or without a 5 min preincubation)were incubatedat 37’C prior to the addition of the agonists.Aliquots were put on ice at different time intervals, and labeled IC or anti-CR1 antibodies added, incubated 2 hours on melting ice, centrifuged over an oil cushion, and pellet as well as supematantcounted. Using this technique we have shownthat PMN bindsthe IC mostly (>95%) via C3b receptors(4). Immunocytochemical localization of CR1 on the surface of PMN, as well as the method of quantification, have beendescribedelsewhere(4).
RESULTS
AND DISCUSSION
We showed previously that a direct correlation exists between the size of CR1 clustersand the efficiency of CR1 to bind IC (ie the percentageof bound IC at a given CR1 concentration) (4). This observation was applied to study the action of various agonists susceptibleto be found at inflammatory sites, and that could induce the aggregation of CRL. Any discrepancy betweenthe percentageof IC binding and CR1 expressionwould indicate a changein receptor distribution, i.e clustering. Cells were incubated for various periods of time (up to 30 min) in the presenceor absenceof an agonist, and IC binding aswell asCR1 expressionwere measured.Figure 1 showsthe typical result from such an experiment. The binding of IC correlated directly with the total CR1 number on PMN incubatedat 37°C. Under such conditions, no change in CR1 clustering takesplace (4). This correlation betweenIC binding and CR1 expression was identical for all the agonistsusedto upregulate CRl. Erythrocytes were much more efficient at binding IC, since to bind 50% of IC, 2.5 times lessCR1 were required (4). Identical resultswere obtainedusingdifferent concentrationsof NAP-l/IL-8 (from 10m9 to 10e7M), fMLP (from 10-sto 10e6M) or C5a (lOs M). Longer periods of incubation at 37OCin the presenceof fMLP (up to 2 hours) provided similar results as well. Similarly, PMA, which has been describedto induce aggregation of CR3 receptors at the surface ofhuman PMN (1 l), did not induce any changein IC binding efficiency (from 0.1 rig/ml to 50 rig/ml, not shown). This demonstratesthat none of the substancesand incubating conditionstestedled to a functionally significant reorganizationof CR1 into large clusters. 1204
Vol.
172,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
50
IO i
00 0
1000
2000
3000
CR1 surface expression
(cpm)
Figure Effect of various agonists on IC binding efficiency. PMN were stimulated with (0) GM-CSF (3ng/ml), (-W-) NAP l/IL-8 (lOnM), (4) TNF (5 @ml), and (0) without agonist for 15 min at 37’C, and IC and CR1 expression was determined. Results are representative of at least 3 other experiments.
These functional
results were confirmed
by direct observation
of CR1 surface
distribution. In no instances did we observe a significant increase in CR1 clustering upon in vitro stimulation
with the above mentioned agonists: in none of these situations the
proportion of clusters containing 3 or more particles exceeded 23% of the total particles counted (mean= 19Sf 3.3%, n=6). This result is comparable to those obtained with circulating PMN (maximum of 19%, mean=13.6&5.5%, n=3) . As extravasation is known to stimulate PMN (12), we measured the CR1 expression and immune complexes bindingof exudate PMN, and compared these values to the one obtained with resting, or in vitro activated cells (Table 1). PMN from the exudate had increased CR1 number as compared to circulating PMN. However,
the expression of CR1
on exudated cells could not be further increasedby treatment with fMLP (lO-‘j M) or PMA (1 rig/ml)
(not shown),
responsiveness
contraryto
circulating
is due to desensitization,
subsequent responsiveness
cells. It is unlikely
because exudation
to fMLP (12). However,
that this non-
primes PMN for their
stimulation
by fMLP has been
described to induce ligand-independent internalization and degradation of CR1 (13). These latter observations expressed
suggest that the internal pool of CR1 might have been completely
during extravasation,
and part of it has been internalized
and subject to
intracellular degradation. These results are in agreement with the data of Berger et al. (10) showing
that neutrophils
from inflammatory
sites have enhanced CR1 expression
compared to circulating cells, and that a subsequent stimulation of these cells with fMLP did only slightly increase CRl. The immune adherence efficiency of exudate PMN was not 1205
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
TABLE
RESEARCH
1 EXPERIMENTS
Circulating
PMN
Exudated
b Relative ’
Percentage
4
22
’
CR1
Binding
CR1
Binding
100
IO
100
13
335
25
540
33
480
28
520
32
1100
55
990
40
-
440
and CR1 expression changes
3
_
580
FMLP-stimulated exudated PMN IC binding
’
.e
PMN
FMLP-stimulated circulating PMN
Binding
100
Heat-stimulated d circulating PMN
a
2
1 CRlb
COMMUNICATIONS
604
of PMN from 3 different
in CR1 expression
with respect
patients.
to circulating
PMN
(100%).
of IC bound.
d PMN were stimulated
15 min at 37°C before
the binding
experiments.
e Not done. ’ PMN were
stimulated
by IuM
FMLP
for 15 min at 37°C
before
the binding
experiments.
changed: given similar level of CR1 expression,a comparable IC binding was obtained (i.e. heat- stimulatedvs exudatedPMN, Table 1). As determined by electron microscopy, the amount of CR1 in clusterscontaining 3 or more gold particles increasedsignificantly in exudatedPMN ascomparedto circulating 60
Cluster
% of total gold particles ,n cluster of a qlve” sue Ctrculating Exudated
0
12
3
PMN
a
PMN
a
5
6
4
7
s,ze
l-2
23
>5
86
14
0.1
71
29
1
8
9
IO
Cluster size Figure: Comparaison of the clustering state of CR1 receptors between circulating (+), and exudated (-D), neutrophils. Erythrocytes (+) distribution is given for comparaison w(from ref. 5). Cells were incubated with anti-CR1 Mab El 1 followed by gold-labeled anti-Mouse Ig, and prepared for label fracture. Mean of 3 experiments.
1206
Vol.
172, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
PMN (Fig. 2). Such an increasein clusteringwas not observedfor heat-activated or fMLPstimulatedPMN from the samedonor (not shown), confirming our previous report (4). In none of the situations analysed, clusters containing more than 8 gold particles were observed(more than 5000 particlescounted), andin exudate, lessthan 30% of the particles were in clustersof 3 or more particles.This latter value hasto be comparedto the situation of erythrocytes, where about 50% of the particles form larger clusters (5). Moreover, the distribution of a control erythrocyte population demonstratedclearly that the difference in clustering betweenerythrocytes and PMN becamenoticeableonly for clusterslarger than 5 particles (Fig. 2), which accountedfor up to 11% of the total, ascomparedto a mere 1% ot the total in exudate PMN (Fig. 2, insert). Two hypothesiscould explain that CR1 clusteringof exudatePMN did not correlated with and increaseIC efficiency. First, CR1 have beenpartially digestedin situ by elastase and other enzymes presentin the exudate (lo), while leaving intact the binding site of the monoclonalusedto tag the receptor(it is postulatedthat monoclonalantibodiesagainstCR1 recognize more than one site per receptor (14)); second,our functional IC binding assay was not sensitiveenoughto detect smallchangesin the aggregationstateof CR 1, ashighly efficient binding may require large clusters(ie larger than 5 CRl), such asthosefound on erythrocytes. Supportingthis latter hypothesis,we recently showed(15) that only a portion of the erythrocytes were capable to bind IC, that no more than 12 IC could bind on the samecell, and that on a CR1 basis,IC binding efficiency was higher for erythrocytes bearing high number of CR1 (hence larger clusters (5)) than erythrocytes with low CR1 number. This would imply that only large clusters -containing clusters larger than 5 receptors, as suggestedby Fig. 2- may play a role in immune adherencereactions, a situation certainly not reached in exudated PMN. Recently, Cosio et al (16), using fluorescentbeadsinsteadof IC, cameto the sameconclusions. The above data reinforcesthe view of a quiescentCR1 receptor on circulating PMN, asopposedto the “active” CR1 on erythrocytes. Only a strong and complex stimulus(ie. exudation) may alter CR1 distribution on PMN. Although not sufficient to significantly increasein vitro binding efficiency, this increasedCR1 clustering may affect significantly the in vivo neutrophil’sfunctions suchasphagocytosis. ACKNOWLEDGMENTS We thank Prof. M. Baggiolini for providing us NAP-l/IL-g, and for his helpful reviewing of the manuscript. We acknowledgethe excellent technical assistanceof Miss Gertraud Steiger and Patrice Fruleux. This work was supported by the Swiss National Science Foundation, grants 32-25606.88 and 31-36625.89. J.A.S. is recipient of a Max Clo&ta CareerDevelopmentAward. REFERENCES 1. Fearon D.T. , and Collins L.A. (1983) J. Immunol. 130, 370-375 2. Wright S.D, and Si1versteinS.C.(1983) J. Exp. Med.158, 2016-2023. 1207
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
3. Medof, M.E., Iida, K., Mold C., and Nussenzweig, V. (1982)J. Exp. Med.156, 1739-1754. 4. Paccaud, J.-P., Carpentier, J.-L., and Schifferli, J.A. (1990) Eur. J. Immunol. 20, 283-289. 5. Paccaud, J.-P., Carpentier, J.-L., and Schifferli, J.A. (1988) J. Immunol. 141, 3889-3894. 6. Berger, M., Birx, D.L., Wetzler, E.M., O’Shea, J.J., Brown, E.J., Cross, A.S., (1985) J. Immunol. 135, 1342-1348. 7. Shalit, M., Von Allmen, C., Atkins, P.C., Zweiman, B. (1988) J. Leuk. Biol. 44, 212-217. 8. Paccaud, J.-P., Schifferli, J.A., and Baggiolini, M. (1990) B&hem. Biophys. Res. Comm.166, 187-192. 9. Berger, M, Wetzler, E.M., Wallis, R.S. (1988) Blood 71, 151-158. 10. Berger, M., Sorensen, R.U., Tosi, M.F., Dearborn D.G., Doring G., (1989) J. Clin. Invest. 84 1302-1313. 11. Detmers, P.A., Wright, S.D., Olsen,E.,Kimball, B.,and Cohn, Z.A. (1987) J. Cell. Biol.105, 1137-l 145 12. Zimmerli, W., Seligmann, B., and Gallin, J.I. (1986) J. Clin. Invest.77, 925933. 13. Turner, J.R., Tartakoff, A.M., and Berger, M. (1988) J. Biol. Chem. 263, 49144920. 14. Bat-tow, T.J., Klickstein , L.B., Fearon, D.T. (1989) Complement Inflamm. 6, 312 (abstract). 15. Madi, N., Paccaud, J.-P., Steiger, G., and Schifferli J.A., (1990) Submitted for publication. 16. Cosio, F.G., Xiao-Ping, S., Hebert, L.A., Clin. Immunol. Immunopathol. 55, 337-354.
1208
Vol. 172, No. 3, 1990 November 15, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1203-1208
EXUDATION INDUCES CLUSTERING OF CR1 RECEPTORS AT THE SURFACE OF HUMAN POLYMORPHONUCLEAR LEUKOCYTES J.-P. Paccaud,J.-L. Carpentier, and J.A. Schifferli* Institute of Histology andEmbryology, University Medical Center, Geneva, *Division of Nephrology, Departmentof Medicine, Geneva, Switzerland Received
September
24,
1990
The complementreceptor type 1 (CRl) surfacedistribution, density and immuneadherence efficiency were determined in circulating PMN activated by fMLP, NAP-l/IL-8, TNF, GM-CSF and C5a, or exudate PMN harvestedfrom skin-blisters.Theseobservationswere comparedwith thoseobservedon restingpet-i-pheralblood PMN. PMN activators known to upregulateCR1 expressiondid not induce a significant increase in CR1 clustering, or immuneadherenceefficiency towardsopsonizedimmunecomplexes. By contrast, increasein CR1 density at the surfaceof exudatedPMN was accompaniedby an increasedclustering. This clustering was however insufficient to increasethe binding efficiency for immune complexes. Eventually, CR1 expression of exudated neutrophil could not be increasedfurther by stimulationwith fMLP or PMA. Theseresults indicated that clustering of CR1 on PMN may occur in vim. Such reaction might determine the phagocytic potential of the cell for opsonized micro-organismsor debris.This clusteringcould not be attributed to one of the PMN activators tested. 0 1990 Ac*demlc Press,Inc. Upon activation, human polymorphonuclear leukocytes (PMN) upregulate complementreceptorstype 1 (CRl, CD35) (l), which are involved in immune adherence reactions, aswell asin phagocytosisof complement-reactedparticles (2,3). However, the efficiency of immune adherencereactionsdoesnot appearto dependon the increasedCR1 density, but rather on the degreeof CR1 clustering. Indeed, in vitro activation of PMN by the chemotactic factor fMLP which increasesthe surface density of CRI, but not the proportion of clusteredreceptors(4), leadsto a poor binding efficiency ascomparedto that of erythrocytes, known to bear clusteredCR1 (5). Several substancessusceptibleto be found at inflammatory sitessuchasC5a, LTB4, PAF (1,6,7), NAP-l/IL-8
(8), or tumor necrosis factor (TNF) (9) are also known to
upregulateCRl. Furthermore, complementreceptorsare increasedon the surfaceof PMN in inflammatory exudates (10). We comparedthe surface distribution of CR1 in exudate and peripheral blood PMN and showthat migration into an exudate leadsto an increasein CR1 clustering, increasethat could not be mimicked in vitro by severalknown upregulators of CRl. METHODS Buffers and reagents. Ficoll 400 and Dextran T500 were obtained from Pharmacia.Bovine serum albumine (BSA), N-formyl-methionyl-leucyl-phenylalanine 0006-291
1203
Xi90
$1.50
Vol.
172, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
(fMLP), phorbol meristate acetate (PMA) from Sigma. Purified human C5a was kindly provided by Dr. Dahinden, Institute of Clinical Immunology, University of Bern. Human recombinant Tumor NecrosisFactor (TNF) was a gift from Dr. G. Grau, Departmentof Pathology, University Medical Center, Geneva. Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) was kindly provided by Dr. J.-J. Mermod, Glaxo, Geneva. [125-1]radiolabeled monoclonalantibody El 1 directed againstCR1 was usedto quantitate CR1 sites on PMN (4). Complement-reacted Hepatitis B surface antigen immune complexes (IC) were prepared asdescribed(4). Phosphatebuffer saline,pH 7.4 , as such (PBS), or supplementedwith 5mg/ml BSA (PBSB) wasused. Cell purification. Resting blood PMN were purified at 4’C (4). Exudate PMN were collected asfollowed: after ethanol disinfection, 4 skin blisterswere produced on the thigh by a suction chamber to which a vacuum of -200 mmHg was applied for 3 hours. The top of the blisters were removed and the lesionscovered by a sterile adhesiveplastic film (Opsite). After approximately 15 hours, the fluid under the plastic film was collected with a syringe. Between 5 to 25~10~cells were obtained with this method, 95% of which were PMN. After washingthe exudatein ice cold PBSB, viability was assessed by Trypan Blue exclusion and wasalways greater than 97%. CR1 measurement and IC binding assays were performed as previously described(4). Briefly, cells (with or without a 5 min preincubation)were incubatedat 37’C prior to the addition of the agonists.Aliquots were put on ice at different time intervals, and labeled IC or anti-CR1 antibodies added, incubated 2 hours on melting ice, centrifuged over an oil cushion, and pellet as well as supematantcounted. Using this technique we have shownthat PMN bindsthe IC mostly (>95%) via C3b receptors(4). Immunocytochemical localization of CR1 on the surface of PMN, as well as the method of quantification, have beendescribedelsewhere(4).
RESULTS
AND DISCUSSION
We showed previously that a direct correlation exists between the size of CR1 clustersand the efficiency of CR1 to bind IC (ie the percentageof bound IC at a given CR1 concentration) (4). This observation was applied to study the action of various agonists susceptibleto be found at inflammatory sites, and that could induce the aggregation of CRL. Any discrepancy betweenthe percentageof IC binding and CR1 expressionwould indicate a changein receptor distribution, i.e clustering. Cells were incubated for various periods of time (up to 30 min) in the presenceor absenceof an agonist, and IC binding aswell asCR1 expressionwere measured.Figure 1 showsthe typical result from such an experiment. The binding of IC correlated directly with the total CR1 number on PMN incubatedat 37°C. Under such conditions, no change in CR1 clustering takesplace (4). This correlation betweenIC binding and CR1 expression was identical for all the agonistsusedto upregulate CRl. Erythrocytes were much more efficient at binding IC, since to bind 50% of IC, 2.5 times lessCR1 were required (4). Identical resultswere obtainedusingdifferent concentrationsof NAP-l/IL-8 (from 10m9 to 10e7M), fMLP (from 10-sto 10e6M) or C5a (lOs M). Longer periods of incubation at 37OCin the presenceof fMLP (up to 2 hours) provided similar results as well. Similarly, PMA, which has been describedto induce aggregation of CR3 receptors at the surface ofhuman PMN (1 l), did not induce any changein IC binding efficiency (from 0.1 rig/ml to 50 rig/ml, not shown). This demonstratesthat none of the substancesand incubating conditionstestedled to a functionally significant reorganizationof CR1 into large clusters. 1204
Vol.
172,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
50
IO i
00 0
1000
2000
3000
CR1 surface expression
(cpm)
Figure Effect of various agonists on IC binding efficiency. PMN were stimulated with (0) GM-CSF (3ng/ml), (-W-) NAP l/IL-8 (lOnM), (4) TNF (5 @ml), and (0) without agonist for 15 min at 37’C, and IC and CR1 expression was determined. Results are representative of at least 3 other experiments.
These functional
results were confirmed
by direct observation
of CR1 surface
distribution. In no instances did we observe a significant increase in CR1 clustering upon in vitro stimulation
with the above mentioned agonists: in none of these situations the
proportion of clusters containing 3 or more particles exceeded 23% of the total particles counted (mean= 19Sf 3.3%, n=6). This result is comparable to those obtained with circulating PMN (maximum of 19%, mean=13.6&5.5%, n=3) . As extravasation is known to stimulate PMN (12), we measured the CR1 expression and immune complexes bindingof exudate PMN, and compared these values to the one obtained with resting, or in vitro activated cells (Table 1). PMN from the exudate had increased CR1 number as compared to circulating PMN. However,
the expression of CR1
on exudated cells could not be further increasedby treatment with fMLP (lO-‘j M) or PMA (1 rig/ml)
(not shown),
responsiveness
contraryto
circulating
is due to desensitization,
subsequent responsiveness
cells. It is unlikely
because exudation
to fMLP (12). However,
that this non-
primes PMN for their
stimulation
by fMLP has been
described to induce ligand-independent internalization and degradation of CR1 (13). These latter observations expressed
suggest that the internal pool of CR1 might have been completely
during extravasation,
and part of it has been internalized
and subject to
intracellular degradation. These results are in agreement with the data of Berger et al. (10) showing
that neutrophils
from inflammatory
sites have enhanced CR1 expression
compared to circulating cells, and that a subsequent stimulation of these cells with fMLP did only slightly increase CRl. The immune adherence efficiency of exudate PMN was not 1205
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
TABLE
RESEARCH
1 EXPERIMENTS
Circulating
PMN
Exudated
b Relative ’
Percentage
4
22
’
CR1
Binding
CR1
Binding
100
IO
100
13
335
25
540
33
480
28
520
32
1100
55
990
40
-
440
and CR1 expression changes
3
_
580
FMLP-stimulated exudated PMN IC binding
’
.e
PMN
FMLP-stimulated circulating PMN
Binding
100
Heat-stimulated d circulating PMN
a
2
1 CRlb
COMMUNICATIONS
604
of PMN from 3 different
in CR1 expression
with respect
patients.
to circulating
PMN
(100%).
of IC bound.
d PMN were stimulated
15 min at 37°C before
the binding
experiments.
e Not done. ’ PMN were
stimulated
by IuM
FMLP
for 15 min at 37°C
before
the binding
experiments.
changed: given similar level of CR1 expression,a comparable IC binding was obtained (i.e. heat- stimulatedvs exudatedPMN, Table 1). As determined by electron microscopy, the amount of CR1 in clusterscontaining 3 or more gold particles increasedsignificantly in exudatedPMN ascomparedto circulating 60
Cluster
% of total gold particles ,n cluster of a qlve” sue Ctrculating Exudated
0
12
3
PMN
a
PMN
a
5
6
4
7
s,ze
l-2
23
>5
86
14
0.1
71
29
1
8
9
IO
Cluster size Figure: Comparaison of the clustering state of CR1 receptors between circulating (+), and exudated (-D), neutrophils. Erythrocytes (+) distribution is given for comparaison w(from ref. 5). Cells were incubated with anti-CR1 Mab El 1 followed by gold-labeled anti-Mouse Ig, and prepared for label fracture. Mean of 3 experiments.
1206
Vol.
172, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
PMN (Fig. 2). Such an increasein clusteringwas not observedfor heat-activated or fMLPstimulatedPMN from the samedonor (not shown), confirming our previous report (4). In none of the situations analysed, clusters containing more than 8 gold particles were observed(more than 5000 particlescounted), andin exudate, lessthan 30% of the particles were in clustersof 3 or more particles.This latter value hasto be comparedto the situation of erythrocytes, where about 50% of the particles form larger clusters (5). Moreover, the distribution of a control erythrocyte population demonstratedclearly that the difference in clustering betweenerythrocytes and PMN becamenoticeableonly for clusterslarger than 5 particles (Fig. 2), which accountedfor up to 11% of the total, ascomparedto a mere 1% ot the total in exudate PMN (Fig. 2, insert). Two hypothesiscould explain that CR1 clusteringof exudatePMN did not correlated with and increaseIC efficiency. First, CR1 have beenpartially digestedin situ by elastase and other enzymes presentin the exudate (lo), while leaving intact the binding site of the monoclonalusedto tag the receptor(it is postulatedthat monoclonalantibodiesagainstCR1 recognize more than one site per receptor (14)); second,our functional IC binding assay was not sensitiveenoughto detect smallchangesin the aggregationstateof CR 1, ashighly efficient binding may require large clusters(ie larger than 5 CRl), such asthosefound on erythrocytes. Supportingthis latter hypothesis,we recently showed(15) that only a portion of the erythrocytes were capable to bind IC, that no more than 12 IC could bind on the samecell, and that on a CR1 basis,IC binding efficiency was higher for erythrocytes bearing high number of CR1 (hence larger clusters (5)) than erythrocytes with low CR1 number. This would imply that only large clusters -containing clusters larger than 5 receptors, as suggestedby Fig. 2- may play a role in immune adherencereactions, a situation certainly not reached in exudated PMN. Recently, Cosio et al (16), using fluorescentbeadsinsteadof IC, cameto the sameconclusions. The above data reinforcesthe view of a quiescentCR1 receptor on circulating PMN, asopposedto the “active” CR1 on erythrocytes. Only a strong and complex stimulus(ie. exudation) may alter CR1 distribution on PMN. Although not sufficient to significantly increasein vitro binding efficiency, this increasedCR1 clustering may affect significantly the in vivo neutrophil’sfunctions suchasphagocytosis. ACKNOWLEDGMENTS We thank Prof. M. Baggiolini for providing us NAP-l/IL-g, and for his helpful reviewing of the manuscript. We acknowledgethe excellent technical assistanceof Miss Gertraud Steiger and Patrice Fruleux. This work was supported by the Swiss National Science Foundation, grants 32-25606.88 and 31-36625.89. J.A.S. is recipient of a Max Clo&ta CareerDevelopmentAward. REFERENCES 1. Fearon D.T. , and Collins L.A. (1983) J. Immunol. 130, 370-375 2. Wright S.D, and Si1versteinS.C.(1983) J. Exp. Med.158, 2016-2023. 1207
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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