The American Journal of PATHOLOGY March 1978
-
Volume 90, Number 3
Chemotactic Factor Influences on the Aggregation, Swelling, and Foreign Surface Adhesiveness of Human Leukocytes J. T. O'Flaherty, MD, D. L. Kreutzer, PhD, and P. A. Ward, MD
Chemotactic factors have been shown to induce aggregation and cellular swelling of rabbit polymorphonuclear neutrophils (PMN-) obtained from the peritoneum. We examined the ability of the chemotactic fragment of C5 and the synthetic chemotactic tripeptide formvl-methionvl-leucvl-phenvlalanine to induce these changes in various preparations of human leukocvtes. Wle found that these factors did induce dextransedimented leukocvtes and Ficoll-Hypaque-isolated PMNI to aggregate and swell. Compared with rabbit peritoneal PMNE, however, human PMNI responded with more prominent swelling but with less prominent aggregation. Also unlike rabbit peritoneal PMNI. human PMN- adhered spontaneouslI- to plastic surfaces: the chemotactic factors enhanced this adherence. Certain similarities between the responses of these two cell ty pes were evident: in both rabbit peritoneal and isolated human peripheral PMIN. the aggregates had a short life span in the fluid phase; in both, the number of aggregates formed was proportional to the log,0 of the PMIN concentration: and. in both, the chemotactic activity of the reagents paralleled their aggregating activity. In the system employed, lymphocytes were unresponsive to the chemotactic factors. Ficoll-Hypaqueisolated monoclear cells (containing -ar-ing proportions of monocytes and lymphocytes) were responsive, indicating that human monocy tes behave in a manner similar to the human PIN. The results suggest that chemotactic factors induce responsive cells to develop a hvperadherent cytoplasmic membrane. Aggregation and increased adhesiseness to plastic surfaces may reflect this induction. (Am J Pathol 90:537-550, 1978)
CHENIOTACTIC FACTORS induce transient aggregation of rabbit peritoneal polymorphonuclear neutrophils (P-N) suspended in phy-siologic media.' In this report wve studied human leukocy-tes and purified populations of PMN, mononuclear cells, and lymphocytes for responses to From the Department of Patholozy. The Universitv of Connectictit School of Mledicine. The 1.ni\ersit\ of Connecticut Health Center. Farmincton. Connectictit Supported in part hb Grants HLOA574 and A11.3910 Accepted for publication October JS. 1977. Address reprint re(quests to Dr Joseph O'Flaherty. Department of Pathology. The Unisersit\ of Connecticut School of Mledicine. The Unisersitv of Connecticut Health Center. Farmincton. CT
06032.
537
538
O'FLAHERTY ET AL
American Journal of Pathology
the chemotactic fragment of C3 (C5 fr) and to the synthetic chemotactic tripeptide formv-l-methionv-l-leucv-l-phenv-lalanine (F-Nt et-Leu-Phe). Leukocytes and PNIN aggregated promptly on exposure to these chemotactins. Lymphocvtes did not respond. Nfononuclear cells containing varving proportions of lymphocytes and monocytes showved significant aggregation, suggesting that monocvtes may behav-e like P\MN. These results confirm and extend a recent report showving that zvmosan-activated plasma and trvpsinized C3 aggregate human PM1N.2 Chemotactic factors may induce PM N (and monocvte) surface membranes to become hyperadherent. Aggregation wvould then ensue. Chemotactic factors also induced prominent swelling of PMN:N this effect w,as independent of aggregation. This cellular swelling, which was considerably greater than that found in rabbit peritoneal PMIN. may reflect the membrane ruffling and pseudopodia formation kno-n to occur ,when these cells are exposed to chemotactins.2'3 PMIN adhered to plastic surfaces spontaneously and the chemotactic factors enhanced this process. This enhanced foreign surface adhesiveness, like aggregation. may reflect increased cx-toplasmic membrane adhesiveness.
Materials and Methods Chemotactic Factors C5 fr Xvas generated from normal human serums and X as purified as previously described. 14.5 F-Met-Leu-Phe X as obtained and stored as previously described 1.5-6
Isolation of Human Blood Cells Leukocvtes were obtained bv sedimentation of human blood (containing both 10 m\1 ethvlenediamine tetraacetic acid and 50 units ml heparin sulfate as anticoagulants) with an equal volume of lox. molecular weight :3%V dextran. After sedimentation for 20 to 30 minutes. the leukocvtes were washed once. hy potonically ly sed three times. and stored at 4 C until use. To obtain purified populations of PMN and mononuclear cells. human \-hole blood collected as above "as layered on Ficoll-Hypaque discontinuous gradients and centrifuged for 20 minutes at 800 x g.7 8 The resulting two bands of cells were separated and treated the same as the dextran-sedimented cells. To obtain purified populations of lymphocytes, the mononuclear laver obtained from Ficoll-Hypaque sedimentation was suspended in regular Hanks' medium (10,000 cells gl) and applied twice to nylon fiber columns containing 100 mg nylon fiber per column. The eluent cells %ere -ashed once before use. Chemotaxis
Both dextran-sedimented leukoctes and Ficoll-Hypaque-isolated PMIN were used in chemotactic assays which employed modified Boyden chambers 9 and Millipore filters of porosity. Chambers were incubated for 45 minutes at 37 C. and chemotaxis was scored :3-p by counting the total number of cells which migrated 10 u or farther into the filter.10
Vol. 90, No. 3 March 1978
CHEMOTACTIC FACTOR INFLUENCES ON LEUKOCYTES
539
The assav to measure aggregation has been previouslv described.1 13 Studies were performed in a 37 C room; reagents were brought to 37 C before use. Cells were obtained as described above, diluted in Hanks' medium, and placed in plastic vials (1 cc suspension per vial). The suspension was continuouslv stirred by the rotations of a magnetic bar. Small volumes of chemotactic factors (50 Ml or less) were added directlv to the suspension and, at various times before and after the addition, 25-pl samples were obtained, diluted in 10 ml of Isoton, and analvzed immediatelv with the Coulter counter svstem. The system consisted of a model ZBI Coulter counter equipped with a mean particle volume computer II and a 100-channel volume channelyzer. The system aspirates 500 yI of the Isoton-diluted suspension and Nields the particle concentration, mean particle volume, and the frequency-volume size distribution of the enumerated specimen. Samples were aspirated through a 100-a aperature slit and the following Coulter counter settings were used: amplification, ¼; aperature, 4; lower and upper thresholds, 10 and infinity, respectively. With these settings the total number of particles was accuratelv recorded and any large particles evolving during the experiment were enumerated with the proper volume sizes. For human cells, better discrimination of aggregation was observed if, in addition to the reading at the lower threshold of 10, another reading of the Isoton-diluted specimen was taken using a lower threshold of 80. At this lower threshold the Coulter counter records only those particles greater than 1.8 times the average PMN size. Thus, the counter enumerates PMN aggregates selectivelv. The number of particles obtained with the counter at this lower threshold of 80 was multiplied by 100 and divided by the total number of particles in the specimen (obtained by enumerating particles at the lower threshold of 10) to obtain the large particle percentage. For comparing various data, the aggregation index (AI) was calculated as follows: AI = Pj/2 + P1 -2 X Po,
where PI 2 and P1 are the percentage of larger particles found 30 and 60 seconds after the addition of the chemotatic factor and Po is the percentage of large particles found less than 30 seconds before the addition. Unless otherwise noted, the cellular concentration for all studies was 4600/Ml and the Hank's medium contained 1.4 mM Ca2+ and 0.7 mM Mg2+, 0.134 mM P04 and 0.74 mNM S04 anions, 5.3 mM1 K+, 200 mg glucose/100 ml, and 25 mM Tris buffer. For chemotactic assays the Hanks' medium contained 500 mg salt-poor human albumin/100 ml Hanks' medium. The albumin did not influence aggregation assavs and was, therefore, omitted. The Hanks' medium and all reagents were brought to pH 7.4 before use.
Results PMN
The PMN populations isolated from Ficoll-Hypaque cushions were greater than 97% pure (mean, 98.4% ± 1.0). When these cells were suspended in bivalent-cation-containing Hanks' medium, the C5 fr induced a fall in particle concentration and a simultaneous rise in mean particle volume (Text-figure 1, solid lines). The effect was sustained over the entire 15-minute observation interval. In Hanks' medium free of bivalent cations, these changes were blunted (Text-figure 1, interrupted lines). F-Met-Leu-Phe induced changes similar to the C5 fr (Table 1).
540
O'FLAHERTY ET AL
American Journal of Pathology
Addition of buffer or human albumin to the PMN suspension -was not associated wNith changes in mean particle volume, although particle concentration fell (Table 1). Identical falls in particle concentration occurred spontaneously. In rabbit peritoneal PMN, chemotactic-factor-induced aggregation is transient and changes in mean particle volume and particle concentration reverse wvithin 8 minutes of adding the chemotactin. Hence, these results suggest that chemotactic factors induce nonrev-ersing aggregation of human PMN. Howvever, further study indicated that this was not the case. WNhen the frequency-volume size distribution of untreated PMN suspended in regular Hanks' medium wvas analyzed wvith a v-olume channelyser. a homogeneous, unimodel peak was seen (Text-figure 2. top). Ninietv seconds after adding C3 fr to these PM N suspensions, the number of cells in the major peak fell and a ne%-. peak wvith a mode volume size twvice that of the major peak appeared (Text-figure 2. center). Eight minutes after the addition. the changes had partially reversed (not showvn). Fifteen minutes after the addition, the changes had returned to baseline (Text-figure 2. bottom). The significant decrease in cell number found in channels 10 to 20 and the significant increase in cell number found in channels :30 to 40 15 minutes after the addition (Text-figure 2. bottom) reflect a rightw%ard shift to larger volume sizes for all the cells in the major peak, ie. each enumerated particle had swvelled slightly. This shift is clearly seen in Text-figure .3 'which plots the channel posit ion for the mode of the major population after adding C5 fr. F-Met-Leu-Phe induced identical changes in both the volume-size distribution and chanPERCENT RISE IN MEAN PARTICLE VOLUME
mg 2
(N )9)
(Nz
11)
TEXT-FI(AHF
(!SE)
chanize
in
1-Percent
mean
particle
%5Alime
dnd particle cin centration fou)tnd at %arious
fr to
PERCENT FALL IN PARTICLE CONCENTRATION (t SE
times
.30,u]
5 AFTER
ADDING C5fr
addingz
C53
suispension suispended in
PM\
Hanks'
mediuim
vvithout
Ca2-
The c-ell
concentration
4600 cells
MINUTES
after
ml o)f
ul
and
vvith
o)r
\1l12> \%as
Vol. 90, No. 3 March 1978
CHEMOTACTIC FACTOR INFLUENCES ON LEUKOCYTES
541
Table 1-Percent Rise in Mean Particle Volume and Percent Fall in Particle Concentration at Various Times After Adding F-Met-Leu-Phe (5 x 10-7 M, Final Concentration), Hanks' Medium, or Human Albumin (12.5 mg/ml, Final Concentration) to Ficoll-Hypaque-isolated PMN Suspensions
Index
Mean particle volume
Time (minutes) 0.25 0.5 1 2 4 8 15
Particle concentration
0.25 0.5 1 2 4 8 15
F-MetLeu-Phe (regular Ca2- and
Mg2-)
F-MetLeu-Phe (no Ca-, no Mg2-)
Hanks' medium
Human albumin
0.1 + 0.4 4.4 ± 0.9 7.7 ± 0.7 8.4 ± 0.7 9.3± 1.1 8.6 0.9 7.4 1.9
0.7 ± 0.7 0.8 ± 0.5 1.8 + 0.4 3.2 ± 0.5 3.4 ± 1.0 3.2 + 0.5 5.7 ± 0.9
-0.4 ± 0.4 -0.1 ± 0.4 -0.1 ±0.4 0.4 ± 0.4 0.4 0.3 0.0 ± 0.6 -0.5 = 1.2
-0.5 ± 0.4 0.2 ± 0.4 0.6 0.4 0.9 ± 0.3 0.8 0.3 1.3 0.6 0.4 + 0.6
2.5 ± 1.1 6.7 ± 1.2 13.2 ± 2.0 13.4 ± 1.7 20.1 ±2.3 18.2 ± 1.8 27.7 3.3
5.2 i 1.7 -1.1 + 2.2 4.3 ± 1.4 3.0 ± 1.8
2.8 ± 0.6 ± 1.6 3.8
2.3 2.2 4.0 2.5
3.0±2.6
5.2±2.1
6.0 ± 2.0 13.5 2.0
5.2 ± 4.1 14.3 5.3
2.4 ± 2.7 2.5 + 2.3 2.1 ±2.6 2.6 ± 2.2 6.2 1.7 3.4 ± 2.2 9.5 1.3
nel position of the major population mode. PMIN suspended in bivalentcation-free Hanks' medium showed neither a fall in the major peak nor the formation of a new peak. although cells in this medium did show a rightwvard shift in the major peak. The shift closely paralleled that in Textfigure 2 (mode in untreated PM1N, 23.75 ± 0.25; mode in PMIN 15 minutes after adding chemotactin, 27.12, P < 0.005). Hence, cellular s,welling, but not aggregation, occurs when PNIN are suspended in bivalent-cation-free media and exposed to chemotactins. The results found by measuring the frequency-volume size distribution (Text-figure 2) conflict with those found by measuring particle concentration and mean particle volume (Text-figure 1). The former measurement suggests that aggregation is transient; the latter suggests it is sustained. In rabbit peritoneal PMIN, both techniques indicate transient aggregation.' By increasing the lower threshold on the Coulter counter to 80 (see Materials and Methods) only particles 1.8 times the mode size of untreated PMIN (corresponding to above Channel 55 in Text-figure 2) are enumerated. Dividing the number of these large particles by the total number of particles in the aspirate and multipl-ing this fraction by 100 yields the percentage of large particles in an aspirate. This modification of the Coulter counter technique allow-s the sequential analysis of the rela-
542
American Journal of Pathology
O'FLAHERTY ET AL H1MAN PAN - FlC(t L bPAd" ISll ATFD
)N OF|
PE
OF PA
ER ADITON | IC FACTCOR (N05)I URED WATh DDIOltON VAWES
10 IN
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TEXT-FHCULRF 2-The frequencyvolume size distribution of P\NI before and at 1.5 and 13 minutes after adding CS fr .500 ml of suspension The Ficoll-Hy paque-isolated P\N m' vere suspended at a c-
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545
546
American Journal of Pathology
O'FLAHERTY ET AL
l2*(N 21) (N- 18)
TEXT-FIGL.RE 5-Percent change in mean particle volume and particle concentration found at various times after adding Co fr (50 Al ml of suspension) to leukocytes suspended in Hanks' medium with or swithout Ca2and \tg2+. The dextransedimented cells sere suspended at a concentration of 4600
MNUTES AFTER ADDNG CSfr
than Ficoll-Hypaque-isolated PMN. With frequent repetition of these experiments, this difference has tended to disappear. Influence of Cell Concentration on Aggregation
Similar to rabbit P\MN,' the magnitude of response of dextran-sedimented leukocy-tes induced by C5 fr wvas proportional to the log1o of the PMN concentration (Text-figure 7). Similar results were found for FMtet-Leu-Phe and for Ficoll-Hvpaque-isolated PMIN (not shown). Correlation of Aggregain and Chemotaxis
Aggregation required higher concentrations of chemotactic factors than did chemotaxis to induce detectable results (Text-figure 8). At the louer OCSfr
(Nf-l
- HUin ALMIN (N'4J _ HANS IEO WI
FEMENT LAMME
0
1
1
MNUTE AFTER
2
4
a
IS
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TEXT-FIcL RE 6-Percentage of large particles found at various times before and after adding 50 Al of CS fr. F-Met-Leu-Phe (5 X 10' NM. final concentration). human albumin, or buffer to 1 ml of leukoc,tes suspended at a concentration of 4600 cells A1 in regular Hanks' medium.
Vol. 90, No. 3 March 1978
CHEMOTACTIC FACTOR INFLUENCES ON LEUKOCYTES
547
Table 4-Percent Rise in Mean Particle Volume and Percent Fall in Particle Concentration at Various Times After Adding F-Met-Leu-Phe (5 x 10' M, Final Concentration), Hanks' Medium, or Human Albumin (12.5 mg/ml, Final Concentration) to Dextran-Sedimented Leukocytes F-Met-Leu-Phe (Regular Ca2- & Index Mean particle volume
Particle concentration
Time
Mg2-)
No Ca2-. no Mg2-
Hanks' medium
Human albumin
0.25 0.5 1 2 4 8 15
1.7 0.6 8.0 ± 1.0 11.8 1.1 11.3 0.9 11.0 0.8 7.9 1.1 4.2 ± 1.3
1.0 + 0.7 0.6 ± 0.3 1.4±0.4 2.0 ± 0.6 2.2 ± 0.4 3.1 ± 0.3 6.8 ± 1.0
0.6 0.7 1.1 ±0.6 1.7±0.8 1.6 0.6 1.4 0.6 0.6 0.6 0.2 ±0.9
0.4 0.4 0.4 ± 0.5 1.7±1.2 1.2 0.5 2.0 0.6 1.1 ±0.8 0.7 ± 1.4
0.25 0.5 1 2 4 8 15
1.7 ± 2.1 18.9 + 3.1 24.0 + 3.0 26.6 ± 2.7 25.8 ± 2.1 22.1 ± 2.1 24.7 ± 2.0
± 4.0 ±2.1 I 3.0 ± 3.8 ± 2.6 4.3 3.5
0.4 ± 2.4 3.1 ± 1.3 6.4 ± 1.9 8.4 + 1.4 8.2 ± 2.9 10.7 1.4 16.6 3.0
-1.6 ± 1.6 4.4-2.4 3.6 ± 1.7 5.1 2.1 5.6 ± 2.0 6.1 2.1 9.2 3.3
2.0 2.1 -0.8 2.9 4.1 6.0 8.9
concentrations of F-Nlet-Leu-Phe, aggregation of leukocytes paralleled chemotaxis. At higher concentrations, chemotaxis plateaued or actualldeclined (Text-figure 4) whereas aggregation continued to increase. These results wvere also found for the C3 fr and for Ficoll-Hypaque-isolated PMN. Rabbit peritoneal PMIN behave similarly.' Discussion Rabbit peritoneal PMIN suspended in physiologic media swvell and transiently aggregate when exposed to chemotactic factors .1"-'3 \'\e 12- HU*iAN LEUKOCYTES DEXTRAN SEDIMENTED
TEXT-FICGIRE 7-Regression line mvith tolerance limits relating the aggregate index to the log10 of leukocyte concentration. C5 fr 50 "I ml of suspension) was added to dextran-sedimented leukocytes suspended at xarious concentrations in regular Hanks merumi. utnrt'- u-4a- aggregate indexes are indicated on the graph.
, /
,0
/
AGGREGATE
INDX 4I
2-
I/I 3
LOGEo
4
PMN CONCENTRATION
.-
,
r
(N 2 4J)
548
O'FLAHERTY ET AL
American Journal of Pathology
HUMAN LE-UKOCYTES 8 - DEXTRAN SED(MENTED
6
'b
-x
AGGREGATE INDEX 4_
/
2-
-
_
__4_________T_____I__I____I__I__I___ 7 T
o
xo10' 1-9
TEXT-FIGc1RE S-The aggre-
- 25 -
e.
2
0
500
,'
T
E8
lo
125
-0
CPER P
H P FF
-
Volume 90, Number 3
Chemotactic Factor Influences on the Aggregation, Swelling, and Foreign Surface Adhesiveness of Human Leukocytes J. T. O'Flaherty, MD, D. L. Kreutzer, PhD, and P. A. Ward, MD
Chemotactic factors have been shown to induce aggregation and cellular swelling of rabbit polymorphonuclear neutrophils (PMN-) obtained from the peritoneum. We examined the ability of the chemotactic fragment of C5 and the synthetic chemotactic tripeptide formvl-methionvl-leucvl-phenvlalanine to induce these changes in various preparations of human leukocvtes. Wle found that these factors did induce dextransedimented leukocvtes and Ficoll-Hypaque-isolated PMNI to aggregate and swell. Compared with rabbit peritoneal PMNE, however, human PMNI responded with more prominent swelling but with less prominent aggregation. Also unlike rabbit peritoneal PMNI. human PMN- adhered spontaneouslI- to plastic surfaces: the chemotactic factors enhanced this adherence. Certain similarities between the responses of these two cell ty pes were evident: in both rabbit peritoneal and isolated human peripheral PMIN. the aggregates had a short life span in the fluid phase; in both, the number of aggregates formed was proportional to the log,0 of the PMIN concentration: and. in both, the chemotactic activity of the reagents paralleled their aggregating activity. In the system employed, lymphocytes were unresponsive to the chemotactic factors. Ficoll-Hypaqueisolated monoclear cells (containing -ar-ing proportions of monocytes and lymphocytes) were responsive, indicating that human monocy tes behave in a manner similar to the human PIN. The results suggest that chemotactic factors induce responsive cells to develop a hvperadherent cytoplasmic membrane. Aggregation and increased adhesiseness to plastic surfaces may reflect this induction. (Am J Pathol 90:537-550, 1978)
CHENIOTACTIC FACTORS induce transient aggregation of rabbit peritoneal polymorphonuclear neutrophils (P-N) suspended in phy-siologic media.' In this report wve studied human leukocy-tes and purified populations of PMN, mononuclear cells, and lymphocytes for responses to From the Department of Patholozy. The Universitv of Connectictit School of Mledicine. The 1.ni\ersit\ of Connecticut Health Center. Farmincton. Connectictit Supported in part hb Grants HLOA574 and A11.3910 Accepted for publication October JS. 1977. Address reprint re(quests to Dr Joseph O'Flaherty. Department of Pathology. The Unisersit\ of Connecticut School of Mledicine. The Unisersitv of Connecticut Health Center. Farmincton. CT
06032.
537
538
O'FLAHERTY ET AL
American Journal of Pathology
the chemotactic fragment of C3 (C5 fr) and to the synthetic chemotactic tripeptide formv-l-methionv-l-leucv-l-phenv-lalanine (F-Nt et-Leu-Phe). Leukocytes and PNIN aggregated promptly on exposure to these chemotactins. Lymphocvtes did not respond. Nfononuclear cells containing varving proportions of lymphocytes and monocytes showved significant aggregation, suggesting that monocvtes may behav-e like P\MN. These results confirm and extend a recent report showving that zvmosan-activated plasma and trvpsinized C3 aggregate human PM1N.2 Chemotactic factors may induce PM N (and monocvte) surface membranes to become hyperadherent. Aggregation wvould then ensue. Chemotactic factors also induced prominent swelling of PMN:N this effect w,as independent of aggregation. This cellular swelling, which was considerably greater than that found in rabbit peritoneal PMIN. may reflect the membrane ruffling and pseudopodia formation kno-n to occur ,when these cells are exposed to chemotactins.2'3 PMIN adhered to plastic surfaces spontaneously and the chemotactic factors enhanced this process. This enhanced foreign surface adhesiveness, like aggregation. may reflect increased cx-toplasmic membrane adhesiveness.
Materials and Methods Chemotactic Factors C5 fr Xvas generated from normal human serums and X as purified as previously described. 14.5 F-Met-Leu-Phe X as obtained and stored as previously described 1.5-6
Isolation of Human Blood Cells Leukocvtes were obtained bv sedimentation of human blood (containing both 10 m\1 ethvlenediamine tetraacetic acid and 50 units ml heparin sulfate as anticoagulants) with an equal volume of lox. molecular weight :3%V dextran. After sedimentation for 20 to 30 minutes. the leukocvtes were washed once. hy potonically ly sed three times. and stored at 4 C until use. To obtain purified populations of PMN and mononuclear cells. human \-hole blood collected as above "as layered on Ficoll-Hypaque discontinuous gradients and centrifuged for 20 minutes at 800 x g.7 8 The resulting two bands of cells were separated and treated the same as the dextran-sedimented cells. To obtain purified populations of lymphocytes, the mononuclear laver obtained from Ficoll-Hypaque sedimentation was suspended in regular Hanks' medium (10,000 cells gl) and applied twice to nylon fiber columns containing 100 mg nylon fiber per column. The eluent cells %ere -ashed once before use. Chemotaxis
Both dextran-sedimented leukoctes and Ficoll-Hypaque-isolated PMIN were used in chemotactic assays which employed modified Boyden chambers 9 and Millipore filters of porosity. Chambers were incubated for 45 minutes at 37 C. and chemotaxis was scored :3-p by counting the total number of cells which migrated 10 u or farther into the filter.10
Vol. 90, No. 3 March 1978
CHEMOTACTIC FACTOR INFLUENCES ON LEUKOCYTES
539
The assav to measure aggregation has been previouslv described.1 13 Studies were performed in a 37 C room; reagents were brought to 37 C before use. Cells were obtained as described above, diluted in Hanks' medium, and placed in plastic vials (1 cc suspension per vial). The suspension was continuouslv stirred by the rotations of a magnetic bar. Small volumes of chemotactic factors (50 Ml or less) were added directlv to the suspension and, at various times before and after the addition, 25-pl samples were obtained, diluted in 10 ml of Isoton, and analvzed immediatelv with the Coulter counter svstem. The system consisted of a model ZBI Coulter counter equipped with a mean particle volume computer II and a 100-channel volume channelyzer. The system aspirates 500 yI of the Isoton-diluted suspension and Nields the particle concentration, mean particle volume, and the frequency-volume size distribution of the enumerated specimen. Samples were aspirated through a 100-a aperature slit and the following Coulter counter settings were used: amplification, ¼; aperature, 4; lower and upper thresholds, 10 and infinity, respectively. With these settings the total number of particles was accuratelv recorded and any large particles evolving during the experiment were enumerated with the proper volume sizes. For human cells, better discrimination of aggregation was observed if, in addition to the reading at the lower threshold of 10, another reading of the Isoton-diluted specimen was taken using a lower threshold of 80. At this lower threshold the Coulter counter records only those particles greater than 1.8 times the average PMN size. Thus, the counter enumerates PMN aggregates selectivelv. The number of particles obtained with the counter at this lower threshold of 80 was multiplied by 100 and divided by the total number of particles in the specimen (obtained by enumerating particles at the lower threshold of 10) to obtain the large particle percentage. For comparing various data, the aggregation index (AI) was calculated as follows: AI = Pj/2 + P1 -2 X Po,
where PI 2 and P1 are the percentage of larger particles found 30 and 60 seconds after the addition of the chemotatic factor and Po is the percentage of large particles found less than 30 seconds before the addition. Unless otherwise noted, the cellular concentration for all studies was 4600/Ml and the Hank's medium contained 1.4 mM Ca2+ and 0.7 mM Mg2+, 0.134 mM P04 and 0.74 mNM S04 anions, 5.3 mM1 K+, 200 mg glucose/100 ml, and 25 mM Tris buffer. For chemotactic assays the Hanks' medium contained 500 mg salt-poor human albumin/100 ml Hanks' medium. The albumin did not influence aggregation assavs and was, therefore, omitted. The Hanks' medium and all reagents were brought to pH 7.4 before use.
Results PMN
The PMN populations isolated from Ficoll-Hypaque cushions were greater than 97% pure (mean, 98.4% ± 1.0). When these cells were suspended in bivalent-cation-containing Hanks' medium, the C5 fr induced a fall in particle concentration and a simultaneous rise in mean particle volume (Text-figure 1, solid lines). The effect was sustained over the entire 15-minute observation interval. In Hanks' medium free of bivalent cations, these changes were blunted (Text-figure 1, interrupted lines). F-Met-Leu-Phe induced changes similar to the C5 fr (Table 1).
540
O'FLAHERTY ET AL
American Journal of Pathology
Addition of buffer or human albumin to the PMN suspension -was not associated wNith changes in mean particle volume, although particle concentration fell (Table 1). Identical falls in particle concentration occurred spontaneously. In rabbit peritoneal PMN, chemotactic-factor-induced aggregation is transient and changes in mean particle volume and particle concentration reverse wvithin 8 minutes of adding the chemotactin. Hence, these results suggest that chemotactic factors induce nonrev-ersing aggregation of human PMN. Howvever, further study indicated that this was not the case. WNhen the frequency-volume size distribution of untreated PMN suspended in regular Hanks' medium wvas analyzed wvith a v-olume channelyser. a homogeneous, unimodel peak was seen (Text-figure 2. top). Ninietv seconds after adding C3 fr to these PM N suspensions, the number of cells in the major peak fell and a ne%-. peak wvith a mode volume size twvice that of the major peak appeared (Text-figure 2. center). Eight minutes after the addition. the changes had partially reversed (not showvn). Fifteen minutes after the addition, the changes had returned to baseline (Text-figure 2. bottom). The significant decrease in cell number found in channels 10 to 20 and the significant increase in cell number found in channels :30 to 40 15 minutes after the addition (Text-figure 2. bottom) reflect a rightw%ard shift to larger volume sizes for all the cells in the major peak, ie. each enumerated particle had swvelled slightly. This shift is clearly seen in Text-figure .3 'which plots the channel posit ion for the mode of the major population after adding C5 fr. F-Met-Leu-Phe induced identical changes in both the volume-size distribution and chanPERCENT RISE IN MEAN PARTICLE VOLUME
mg 2
(N )9)
(Nz
11)
TEXT-FI(AHF
(!SE)
chanize
in
1-Percent
mean
particle
%5Alime
dnd particle cin centration fou)tnd at %arious
fr to
PERCENT FALL IN PARTICLE CONCENTRATION (t SE
times
.30,u]
5 AFTER
ADDING C5fr
addingz
C53
suispension suispended in
PM\
Hanks'
mediuim
vvithout
Ca2-
The c-ell
concentration
4600 cells
MINUTES
after
ml o)f
ul
and
vvith
o)r
\1l12> \%as
Vol. 90, No. 3 March 1978
CHEMOTACTIC FACTOR INFLUENCES ON LEUKOCYTES
541
Table 1-Percent Rise in Mean Particle Volume and Percent Fall in Particle Concentration at Various Times After Adding F-Met-Leu-Phe (5 x 10-7 M, Final Concentration), Hanks' Medium, or Human Albumin (12.5 mg/ml, Final Concentration) to Ficoll-Hypaque-isolated PMN Suspensions
Index
Mean particle volume
Time (minutes) 0.25 0.5 1 2 4 8 15
Particle concentration
0.25 0.5 1 2 4 8 15
F-MetLeu-Phe (regular Ca2- and
Mg2-)
F-MetLeu-Phe (no Ca-, no Mg2-)
Hanks' medium
Human albumin
0.1 + 0.4 4.4 ± 0.9 7.7 ± 0.7 8.4 ± 0.7 9.3± 1.1 8.6 0.9 7.4 1.9
0.7 ± 0.7 0.8 ± 0.5 1.8 + 0.4 3.2 ± 0.5 3.4 ± 1.0 3.2 + 0.5 5.7 ± 0.9
-0.4 ± 0.4 -0.1 ± 0.4 -0.1 ±0.4 0.4 ± 0.4 0.4 0.3 0.0 ± 0.6 -0.5 = 1.2
-0.5 ± 0.4 0.2 ± 0.4 0.6 0.4 0.9 ± 0.3 0.8 0.3 1.3 0.6 0.4 + 0.6
2.5 ± 1.1 6.7 ± 1.2 13.2 ± 2.0 13.4 ± 1.7 20.1 ±2.3 18.2 ± 1.8 27.7 3.3
5.2 i 1.7 -1.1 + 2.2 4.3 ± 1.4 3.0 ± 1.8
2.8 ± 0.6 ± 1.6 3.8
2.3 2.2 4.0 2.5
3.0±2.6
5.2±2.1
6.0 ± 2.0 13.5 2.0
5.2 ± 4.1 14.3 5.3
2.4 ± 2.7 2.5 + 2.3 2.1 ±2.6 2.6 ± 2.2 6.2 1.7 3.4 ± 2.2 9.5 1.3
nel position of the major population mode. PMIN suspended in bivalentcation-free Hanks' medium showed neither a fall in the major peak nor the formation of a new peak. although cells in this medium did show a rightwvard shift in the major peak. The shift closely paralleled that in Textfigure 2 (mode in untreated PM1N, 23.75 ± 0.25; mode in PMIN 15 minutes after adding chemotactin, 27.12, P < 0.005). Hence, cellular s,welling, but not aggregation, occurs when PNIN are suspended in bivalent-cation-free media and exposed to chemotactins. The results found by measuring the frequency-volume size distribution (Text-figure 2) conflict with those found by measuring particle concentration and mean particle volume (Text-figure 1). The former measurement suggests that aggregation is transient; the latter suggests it is sustained. In rabbit peritoneal PMIN, both techniques indicate transient aggregation.' By increasing the lower threshold on the Coulter counter to 80 (see Materials and Methods) only particles 1.8 times the mode size of untreated PMIN (corresponding to above Channel 55 in Text-figure 2) are enumerated. Dividing the number of these large particles by the total number of particles in the aspirate and multipl-ing this fraction by 100 yields the percentage of large particles in an aspirate. This modification of the Coulter counter technique allow-s the sequential analysis of the rela-
542
American Journal of Pathology
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546
American Journal of Pathology
O'FLAHERTY ET AL
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TEXT-FIGL.RE 5-Percent change in mean particle volume and particle concentration found at various times after adding Co fr (50 Al ml of suspension) to leukocytes suspended in Hanks' medium with or swithout Ca2and \tg2+. The dextransedimented cells sere suspended at a concentration of 4600
MNUTES AFTER ADDNG CSfr
than Ficoll-Hypaque-isolated PMN. With frequent repetition of these experiments, this difference has tended to disappear. Influence of Cell Concentration on Aggregation
Similar to rabbit P\MN,' the magnitude of response of dextran-sedimented leukocy-tes induced by C5 fr wvas proportional to the log1o of the PMN concentration (Text-figure 7). Similar results were found for FMtet-Leu-Phe and for Ficoll-Hvpaque-isolated PMIN (not shown). Correlation of Aggregain and Chemotaxis
Aggregation required higher concentrations of chemotactic factors than did chemotaxis to induce detectable results (Text-figure 8). At the louer OCSfr
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TEXT-FIcL RE 6-Percentage of large particles found at various times before and after adding 50 Al of CS fr. F-Met-Leu-Phe (5 X 10' NM. final concentration). human albumin, or buffer to 1 ml of leukoc,tes suspended at a concentration of 4600 cells A1 in regular Hanks' medium.
Vol. 90, No. 3 March 1978
CHEMOTACTIC FACTOR INFLUENCES ON LEUKOCYTES
547
Table 4-Percent Rise in Mean Particle Volume and Percent Fall in Particle Concentration at Various Times After Adding F-Met-Leu-Phe (5 x 10' M, Final Concentration), Hanks' Medium, or Human Albumin (12.5 mg/ml, Final Concentration) to Dextran-Sedimented Leukocytes F-Met-Leu-Phe (Regular Ca2- & Index Mean particle volume
Particle concentration
Time
Mg2-)
No Ca2-. no Mg2-
Hanks' medium
Human albumin
0.25 0.5 1 2 4 8 15
1.7 0.6 8.0 ± 1.0 11.8 1.1 11.3 0.9 11.0 0.8 7.9 1.1 4.2 ± 1.3
1.0 + 0.7 0.6 ± 0.3 1.4±0.4 2.0 ± 0.6 2.2 ± 0.4 3.1 ± 0.3 6.8 ± 1.0
0.6 0.7 1.1 ±0.6 1.7±0.8 1.6 0.6 1.4 0.6 0.6 0.6 0.2 ±0.9
0.4 0.4 0.4 ± 0.5 1.7±1.2 1.2 0.5 2.0 0.6 1.1 ±0.8 0.7 ± 1.4
0.25 0.5 1 2 4 8 15
1.7 ± 2.1 18.9 + 3.1 24.0 + 3.0 26.6 ± 2.7 25.8 ± 2.1 22.1 ± 2.1 24.7 ± 2.0
± 4.0 ±2.1 I 3.0 ± 3.8 ± 2.6 4.3 3.5
0.4 ± 2.4 3.1 ± 1.3 6.4 ± 1.9 8.4 + 1.4 8.2 ± 2.9 10.7 1.4 16.6 3.0
-1.6 ± 1.6 4.4-2.4 3.6 ± 1.7 5.1 2.1 5.6 ± 2.0 6.1 2.1 9.2 3.3
2.0 2.1 -0.8 2.9 4.1 6.0 8.9
concentrations of F-Nlet-Leu-Phe, aggregation of leukocytes paralleled chemotaxis. At higher concentrations, chemotaxis plateaued or actualldeclined (Text-figure 4) whereas aggregation continued to increase. These results wvere also found for the C3 fr and for Ficoll-Hypaque-isolated PMN. Rabbit peritoneal PMIN behave similarly.' Discussion Rabbit peritoneal PMIN suspended in physiologic media swvell and transiently aggregate when exposed to chemotactic factors .1"-'3 \'\e 12- HU*iAN LEUKOCYTES DEXTRAN SEDIMENTED
TEXT-FICGIRE 7-Regression line mvith tolerance limits relating the aggregate index to the log10 of leukocyte concentration. C5 fr 50 "I ml of suspension) was added to dextran-sedimented leukocytes suspended at xarious concentrations in regular Hanks merumi. utnrt'- u-4a- aggregate indexes are indicated on the graph.
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548
O'FLAHERTY ET AL
American Journal of Pathology
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