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Methyl ester of n-formylmethionyl-leucyl-phenylalanine. Chemotactic responses of human blood monocytes and inhibition of gold compounds

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133
METHYL ESTER OF
N-FORMYLMETHIONYLLEUCYL-PHENYLALANINE
CHEMOTACTIC RESPONSES OF HUMAN BLOOD MONOCYTES
AND INHIBITION OF GOLD COMPOUNDS
PETER P. K. HO, ANNA L. YOUNG, and GEORGE L. SOUTHARD
N-formylmethionyl-leucyl-phenylalanine,a potent
chemotactic peptide for human polymorphonuclear leukocytes, is less chemotactic for human blood monocytes.
Esterification of the N-formylated tripeptide enhances its
chemotactic activity for monocytes by more than 4 logs,
whereas a decrease by 3 logs is observed for polymorphonuclear leukocytes. These results indicate the participation of the C-terminal carboxyl group in chemotaxis
of different cell types. We have also observed the selective
inhibition of chemotactic responsiveness of human blood
monocytes by a clinically useful antirheumatic drug, sodium aurothiomalate. Since this is the first in vitro cell
migration model of inflammation in which gold compounds
have demonstrated activity in micromolar concentrations,
it suggests a site of action for this antirheumatic drug.
chemotactic activity, lysosomal enzyme-releasing ability
in rabbit neutrophils (2), histamine-releasing ability in
human basophils (3), and receptor binding on rabbit
neutrophils (4). However, it is not clear whether these
tripeptides are equally active in chemotaxis against both
human polymorphonuclear leukocytes (PMN) and
blood monocytes (MNC). We are. now reporting that
the formylated tripeptides are poor chemoattractants
for M N C and that esterification of the formylated tripeptides increases their chemotactic activity against
MNC by more than four orders of magnitude. In addition, several antiinflammatory and antirheumatic drugs
were examined for their inhibitory activity in chemotaxis of PMN and MNC.
In 1975 Schiffman et al. reported that a number
of small synthetic N-formylmethionyl peptides were
strongly chemotactic for both neutrophils and macrophages (1). They subsequently found that the N-formyl
tripeptides were more effectivc: than dipeptides in their
MATERIALS AND METHODS
From Lilly Research Laboratories, Eli Lilly and Company,
Indianapolis, Indiana 46206.
Peter P. K. Ho, Ph.D.: Immunology, Connective Tissue, and
Physiological Research Administration; Anna L. Young, B.S.:Department of Immunology and Connective Tissue Research; Gary L.
Southard, Ph.D., Department of Biochemistry.
Address reprint requests to Peter P.K. Ho, Ph.D., MC860
Lilly Research Laboratories, Eli Lilly and Company, P. 0. Box 618,
Indianapolis, I N 46206.
Submitted for publication July 25; accepted August 15, 1977.
Arthritis and Rheumatism, Vol. 21, No. 1 (January-February 1978)
N-formylmethionyl-leucyl-phenylalanine(FM LP) was
obtained from Bachem (Marina Del Rey. California). The
methyl ester of F M L P (FMLP-M) was synthesized by routine
19.4 (C =
methods of peptide synthesis; M P 132-135'; [ylDZh0.5, DMF); C,H,S elemental analysis within 0.3% of theory:
chromatographically pure in two standard solvent systems for
peptides (5). Sodium aurothiomalate was obtained from Aldrich Chemicals and sodium aurothioglucose from Sigma
Chemicals.
Blood was drawn in sterile syringes with 0.38% of
sodium citrate/ml of blood from healthy human volunteers
who had not ingested any antiinflammatory drugs including
aspirin for at least 7 days. After low-speed centrifugation (200
X g for 15 minutes), the platelet-enriched plasma was removed
and the cells were resuspended in Dulbecco's phosphate-buffered saline (Grand Island Biological Co, Grand Island, New
HO ET AL
134
York) to two times the original volume of the whole blood.
Purified populations of PMN and mononuclear cells (24%
monocytes and 75% lymphocytes) were obtained by Lymphoprep (Gallard-Schlesinger Chemical Co, Long Island, New
York) flotation (6). In vitro migration of human PMN and
MNC was assayed by a modification (2) of the method described by Boyden (7). The number of cells counted in ten
fields at a magnification of 1250 was used as an index of
chemotactic activity. The chambers were run in duplicate for
each substance.
Deactivation of Cells. The techniques followed those
described by Wasserman et ul. (8) for eosinophils. Monocytes,
at the concentrations to be used for the chemotactic assay,
were incubated with lo-' M FMLP-M at 37°C for the time
specified. The sample was then centrifuged at 200 X g, washed
twice, resuspended in the media, placed above the filter in the
chemotactic chamber, and assayed for cell migration toward
lo-" M FMLP-M. In each experiment an aliquot of cells,
handled exactly as cells exposed to FMLP-M for deactivation,
was suspended in medium only and served as a positive control.
Table 2. Chemotactic Activity of FMLP-M on Human Blood PMN
and MNC*
Molar Concentration
of FMLP-M
Human Blood
PMN
Human Blood
MNC
618 f 35
582 f 35
335 f 27
352 f 27
360 f 25
-
122 f 9
1 7 0 f 15
185 f 15
197 f 10
I30 f 9
110 f 10
22 f 4
Below the filter
10-8
10-7
10-8
10-9
10-10
10-11
10-12
-
Above and below the filter
10-7
10-8
10-9
Casein (5 mg/ml)
Negative control
(Hank's medium)
644 & 30
33 f 6
48 f 8
48 f 7
174 f 13
250 f 38
12f3
-
* The
experimental conditions were identical to those described in
Table 1.
RESULTS
Although Showell et al. (2) have clearly demonstrated by the method of Zigmond and Hirsch (9) that
F M L P is truly chemotactic for rabbit peritoneal P M N ,
Table 1. Chemotactic Activity of FMLP on Human Blood PMN and
MNC*
~
Molar Concentration
of FMLP
Below the filter
lo-'
10-6
10-8
10-7
10-8
10-9
10-10
Above and below the filter
lo-'
10-8
Casein (5 mg/ml)
Negativecontrol (Hank'smedium)
Human Blood
PMN
Human Blood
MNC
-
86f7
38 f 6
I2f2
20 f 6
12f3
517 f 35
490 f 35
732 f 41
645 f 21
416 f 34
155 f 17
704 f 95
105 f 15
28 f 4
91 f 8
14 f 2
*Human blood PMN and MNC were washed and suspended in
Hank's balanced salt solution containing 0.01 M Tris (hydroxymethyl)
amino methane, pH 7.2, 1 mg/ml of glucose and 1 mg/ml of crystalline human serum albumin (Sigma Chemicals). Two-tenths milliliter
purified cells containing 2 X I @ cells/ml was added to the upper
compartment of the modified Boyden Chamber (Bio-Rad Lab)
separated from 0.2 ml of peptide solution by a 13 mm diameter filter
of a 3.0 p m (for PMN) or 5.0 pM (for MNC) average pore size (Unipore membranes, Bio-Rad Lab). The time of incubation was 90
minutes. Numbers quoted are mean chemotactic activity (number of
cells counted in ten fields) of the duplicate.
this finding was confirmed with human P M N and M N C
(Table I ) . When F M L P (lo-" M for P M N and lO-'M
for M N C ) was placed in the lower chamber, a six- t o
sevenfold increase in chemotactic activity was obtained
over the buffer control reaching the value obtained with
5 mg/ml of casein. If both chambers contained FMLP,
only a slight increase in chemotactic activity was observed. F M L P induced migration of P M N a t a concentration as low as
M , but was effective a t lo-' M
for M N C .
The chemotactic activity of the methyl ester of
F M L P was next examined. In contrast to FMLP, with
FMLP-M, the chemotaxis for P M N was sharply reduced by more than 3 logs, whereas chemotaxis for
M N C was increased by a t least 4 logs (Table 2). It was
also shown that the movements of the cells were the
result of chemotaxis rather than random movement.
A characteristic of any true chemotactic factor is
its ability t o deactivate cells to further chemotaxis by
prior incubation. This is also considered a criterion for
its cell specificity (8).
Deactivation of monocytes t o FMLP-M was
demonstrated by first exposing the cells to the chemotactic agent for varying time intervals and then, after
washing, utilizing these cells for the chemotactic assay
with FMLP-M at the same concentration used for deactivation.
Compared to cells exposed to medium only
(chemotactic activity of 424 f 42 for FMLP-M and 37
f 8 for negative control), monocytes became unresponsive t o further directional stimulation after 15 to 30
FMLP AND GOLD COMPOUNDS
minutes of incubation with FMLP-M (chemotactic activity of 81 f 21 and 49 f 8 after I5 and 30 minutes of
incubation, respectively).
Neither indomethacin, a nonsteroidal antiinflammatory drug, nor gold compounds (such as sodium aurothiomalate and sodium aurothioglucose) inhibited PMN chemotaxis at a concentration of
M
utilizing FMLP as the chemotactic agent. In contrast,
gold compound inhibited MNC chemotaxis in a doseresponse manner (Figure 1 ), whereas indomethacin inhibited slightly (30-50% at lO-'M). The I,, values for
these drugs were: sodium aurothiomalate 4 X lo-' M ;
and sodium aurothioglucose, 1 X
M . There is little
difference in the values of I,, when lo-' M FMLP,
M FMLP-M, or casein ( 5 mg/ml) is employed as the
chemotactic agent.
Furthermore, preliminary experiments indicate
that inhibition of chemotaxis by gold compounds probably acts via competition against the binding of FMLPM to the cells. Table 3 shows that decreasing the concentration of FMLP-M increases the percentage of inhibition by a fixed concentration of aurothiomalate.
DISCUSSION
In addition to the structure requirement for specific amino acids and acylation of the N-terminal amino
group as reported by Showell et al. (2), we have discovered the participation of the C-terminal carboxyl group
in the chemotaxis of PMN. Of particular interest is the
finding that the methyl ester of FMLP is a potent chemotactic agent for monocytes. This suggests that although
there is a common structural requirement (that is, the
formylated peptide) for the receptor sites of PMN and
monocytes, the C-terminal carboxyl group determines
the preference of binding to one cell type over the other.
I n view of the report by Aswanikumar et al. (4) that a
specific chemotactic receptor exists on rabbit neutrophil
membranes, it would be interesting to see whether similar receptors also exist on human blood monocyte
membranes that mediate chemotaxis induced by FMLPM.
Several mechanisms have been proposed to explain the actions of gold compounds in rheumatoid
arthritis. These include inhibition of the lysosomal enzymes of the peritoneal macrophage (lo), human granulocyte elastase ( 1 I ), human leukocyte collagenase
( I 2,13), and breakdown of noncollagenous chondromucoprotein matrix by leukocyte lysosome granule lysates
from guinea pigs (14). More recently, sodium aurothiomalate was shown to inhibit antigen and mitogen-
I35
20-
3
4
SOD1UM
5
6
7
8
9
AUROTH IOMALATE
( -LOG MOLAR )
Figure 1. Efect of various concentrations ofgold compounds on the chemotactic responsiveness of human blood M N C . Chemotaxis was measured
as described in Table I . Gold compounds were mixed with the cells and
IO-'M F M L P - M was employed as the chemotactic agent. Variations
were from two separate experiments. The mean chemotactic activity of
the duplicate was 146 f 20 for FMLP-M control and 21 f 3 for
negative control.
induced human lymphocyte proliferation (15-1 7). However, these effects were observed at relatively high,
though probably achievable in vivo, concentrations
(l0-l to
M ) of gold compounds.
In rheumatoid patients, sodium aurothiolamate
was found to inhibit the cellular and fluid phase of the
inflammatory response and suppress the phagocytic activity of inflammatory macrophages and PMN ( 1 8). In
addition, sodium aurothiomalate also inhibited the in
Table 3. Efect of Chemoractic Factor Concentration on Inhibition
Concentration of
FMLP-M ( M )
10-1
10-6
10-0
10-'0
Percent Inhibition with
lo-' M Aurothiomalate
41
62
83
100
HO ET AL
136
vitro migration of exudate macrophages isolated from
drug-treated rats by the capillary tube migration method
( 1 9).
The present report, therefore, substantiates this
early observation by Vernon-Roberts that gold compounds have a profound effect on inflammatory cells
(19). The most interesting finding of our data is the
selective inhibition of gold compounds on MNC chemotaxis. In contrast, Ward et al. (20) reported the suppressive effect of metal salts on neutrophil chemotactic function. However, the concentration required for its
inhibition was approximately low4M gold chloride.
The fact that two different classes of clinically
useful drugs act differently in the in vitro models further
suggests the multiple actions of antirheumatic drugs.
The mechanism of action of aspirin-like drugs, such as
indomethacin, has been thought to be mediated through
their inhibition on fatty acid cyclo-oxygenase, although
it was observed in the present experiments that indomethacin also slightly inhibits chemotaxis of MNC
(30-5096 at lo-' M ) at 100 times the concentration required to inhibit the cyclo-oxygenase. On the contrary,
gold compounds do not inhibit the cyclo-oxygenase (21)
but are potent inhibitors of chemotaxis of MNC.
It is obvious, however, that before a definitive
statement can be made with regard to the relationship
between inhibition of MNC chemotaxis and efficacy in
human arthritis and rheumatism, it will be necessary to
have a better understanding of the natural chemotactic
factors and the entire sequence of events that lead to the
migration of monocytes.
REFERENCES
I . Schiffman EB, Corcoran B, Wahl S: N-formyl-methionyl
peptides as chemo-attractants for leukocytes. Proc Natl
Acad Sci USA 72:1059-1062, 1975
2. Showell HJ, -Freer RJ, Freer SH, Zigmond H, et al: The
structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal enzyme secretion for neutrophils. J Exp Med 143:1154-1169, 1976
3. Hook WA, Schiffman E, Aswanikumar S, et al: Histamine
release by chemotactic formyl methionine-containing peptides. J Immunol 117594, 1976
4. Aswanikumar S, Corcoran B, Schiffmann E, et al: Demonstration of a receptor on rabbit neutrophils for chemotactic peptides. Biochem Biophys Res C o m m
74:8 10-8 17, 1977
5. Stewart JM, Young JD: Solid-phase Peptide Synthesis.
San Francisco, W. H. Freeman & CO, 1969
6. Boyum A: Isolation of mononuclear cells and gran-
ulocytes from human blood. Scand J Clin Lab Invest
21:77-89, 1968
7. Boyden S: Chemotactic effect of mixtures of antibody and
antigen on polymorphonuclear leukocytes. J Exp Med
115~453-466, 1968
8. Wasserman SI,Whitmer D , Goetzl El, et al: Chemotactic
deactivation of human eosinophils by the eosinophil
chemotactic factor of anaphylaxis. Proc SOCExp Biol
Med 148:301-306, 1975
9. Zigmond SH, Hirsch JG: Leukocyte locomotion and
chemotaxis. New method for evaluation and demonstration of cell-derived chemotactic factor. J Exp Med
137:387-410, 1973
10. Persellin RH, Ziff M: The effect of gold salt on lysosomal
enzymes of the peritoneal macrophage. Arthritis Rheum
9:57-65, 1966
11. Janoff, A: Inhibition of human granulocyte elastase by
gold sodium thiomalate. Biochem Pharmacol 19:626-628,
I970
12. Wojtecka-Leukasik E, Dancewicz AM: Inhibition of human leukocyte collagenase by some drugs used in the
therapy of rheumatic diseases. Biochem Pharmacol
23:2077-2081, 1974
13. Perper RJ, Oronsky AL: Enzyme release from human
leukocytes and degradation of cartilage matrix. Effects of
antirheumatic drugs. Arthritis Rheum 17:47-55, 1974
14. Ignarro LJ, Oronsky AL, Perper RJ: Breakdown of
noncollagenous chondromucoprotein matrix by leukocyte
lysosome granule lysate from guinea pig, rabbit, and human. Clin Immunol Immunopathol 236-51, 1973
15. Harth M , Stiller CR, Evans J. Zuberi R, et al: Effect of
sodium aurothiomalate on human lymphocyte functions
in normal controls and patients with rheumatoid arthritis.
Arthritis Rheum 19:802, 1976
16. Lipsky PE, Ziff M: Inhibition of antigen- and mitogeninduced human lymphocyte proliferation by gold sodium
thiomalate. Arthritis Rheum 19:808, 1976
17. Panush RS: Effects of certain antirheumatic drugs on normal human peripheral blood lymphocytes. Inhibition of
mitogen- and antigen-stimulated incorporation of tritiated thymidine. Arthritis Rheum 19907-917, 1976
18. Jessop JD, Vernon-Roberts B, Hanis J: Effects of gold
salts and prednisolone on inflammatory cells. I. Phagocytic activity of macrophages and polymorphs in inflammatory exudates studied by a skin-window technique
in rheumatoid and control patients. Ann Rheum Dis
32~294-300, 1973
19. Vernon-Roberts B, Jessop JD, Dore J: Effect of gold salt
and prednisolone on inflammatory cells.' 11. Suppression
of inflammation and phagocytosis in the rat. Ann Rheum
Dis 32:301-307, 1973
20. Ward PA, Goldschmidt P, Greene ND: Suppressive effects of metal salts on leukocyte and fibroblastic function.
J Reticuloendothel SOC18:313-321, 1975
21. H o PPK: Unpublished results
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formylmethionyl, compounds, inhibition, phenylalanine, chemotactic, human, blood, methyl, response, monocyte, esters, leucyl, gold
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