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Modification of Neutrophil Chemotactic Responsiveness During Various Inflammatory Reactions
Path. Res. Pract. 180, 130-135 (1985)
Modification of Neutrophil Chemotactic Responsiveness During Various Inflammatory Reactions M. Roch-Arveiller, A. Perianin, D. Pham Huy and J. P. Giroud (ERA CNRS 629), Department of Pharmacology, 27 rue du Fbg. St-Jacques75014 Paris
SUMMARY Various inflammatory reactions have been induced in order to examine the chemotactic response of polymorphonuclear leucocytes (PMN) collected under various experimental conditions. Cells were harvested from the pleural cavity of rats after the induction of three acute non specific inflammatory reactions and two immune reactions. The results obtained with two techniques of chemotactic assessment (agarose assay and Boyden chamber technique) demonstrated a variation of chemotactic response depending on the cell source and the chemoattractants used. Using agarose assay, we distinguished locomotor reactivity of PMN harvested after immune inflammatory reactions from that of PMN harvested after non immune inflammatory reactions. Chemokinetic and chemotactic responses to various chemoattractants were inhibited in the first case and not affected in the second. Using the Boyden chamber technique, inhibition of random or oriented migration of PMN harvested after immune inflammatory reactions after the injection of a non antigenic irritant such as calcium pyrophosphate crystals (CaPP) was also observed.
Introduction The rapid accumulation of phagocytic wandering cells at sites of inflammation, infection or antigenic penetration appears to be essential for the defence of the host against various injuries. One mechanism which can lead to the local accumulation of motile cells is chemotaxis i.e. the unidirectional migration of cells along a gradient of chemoattractant(s). This parameter must be distinguished from chemokinesis in which sfeed of the cells is modified without change of directions,l , 26, 27. The directed migration of leucocytes in response to chemotactic gradients is a complex series of biochemical This paper is based on a speech held at the IXth European Congress
of Pathology in Hamburg 1983 in the session C 3 "Biochemistry of Inflammation" . 0344-0338/85/0180-0130$3.50/0
phenomena requiring recognition of chemical signals by the cell membrane, the triggering of energy-forming processes within the cell and the translation of this energy by cytostructural elements into movement. The events which result in the directed migration of motile cells as a consequence of their exposure to chemotactic gradients are still unknown. However several lines of evidence suggest that chemotactic factor binding to leucocyte, divalent cation fluxes activation of certain metabolic pathways and polymerisation of actomyosin-like molecules within the cells are required for cell movemenrl,22. Directed migration appears to result from the adaptation of the cells to local changes in stimulus concentration 6, 24. An intact phagocytic system is necessary for normal host defence and a rapid migration into tissues where
surface defences have been breached, is a neutrophil function essential for a good health. These experimental obser© 1985 by Gustav Fischer Verlag, Stuttgart
Modulation of Neutrophil Chemotaxis by Inflammation . 131
vations have been supported recently by several clinical observations which suggest an association between susceptibility to infection and depressed neutrophil chemotactic responsiveness in patients with recurrent severe infections. Numerous reports of intrinsic cellular defects of locomotion in individual patients with serious infections disease problems suggest that this may be a frequent basis for diminished host defence 13 , 17, 18. Defective phagocytic cell locomotion in patients may be the consequence of a variety of causes: - intrinsic cellular defects of locomotion (lazy leucocyte syndrome of Miller 14 and many other syndromes susceptible to exhibit recurrent infections). This defect may be transient in certain patients. - defective generation of chemotactic factors. - inhibition of chemotactic factors described by Mandelll2 in patients with malignancies. The multiple examples of clinical locomotory disorders led us to examine the cellular behaviour of polymorphonuclear leucocytes (PMN) harvested during various experimental inflammatory reactions. Previous studies 19 have demonstrated an inhibition of PMN chemotactic responsiveness during an acute non specific inflammatory reaction (pleurisy after injection of a suspension of CaPP crystals). We want to present here the modification of neutrophil chemotactic response observed after the induction of various inflammatory reactions of non immune and immune origin. Material and Methods
1. Cell Collection Pathogen free, male Sprague-Dawley rats (180-200 g) were used for all experiments. Leucocytes were collected from the pleural cavity 4 hours after the injection of - 1 ml of isologous serum heated fordecomplementation 7, - 1 ml of Phosphate Buffer Saline (PBS), - 1 ml of a suspension of Calcium pyrophosphate crystals (CaPP) (1 %) in saline28 • On the other hand, we have harvested rat PMN after the induction of two immune inflammatory reactions: - a reversed passive Arthus reaction in the pleural cavity29, - a reaction of delayed hypersensitivity on rats sensitized to Bordetella pertussis 4 • The cells were washed twice and resuspended in appropriate medium: Krebs' solution for agarose assays, Hanks' solution with bovine serum albumine (1%) for Boyden chamber technique.
2. Chemotactic assessment It has been performed by two techniques:
A - Migration under agarose
Spontaneous and oriented migration were measured by the modified version of the agarose method15, 16 4 ml of 0,75% (v/v) indubiose A-37 (Industrie biologique fran<;aise - France) in Krebs Ringer Phosphate (KRP) pH 6.8 containing 10% of heat-inactivated foetal calf serum, was poured into small tissue culture Petri
dishes (Falcon 15 X 55 mm - Lab. Express Servo France). Four sets of 3 wells, 2.5 mm in diameter and spaced 2.5 mm apart were cut out using a template; 5 fil of leucocyte suspension (i.e. 5 x 105 PMN) was placed in each of the middle wells (PMN well), 5 fil of chemotactic factors in the outer wells (chemoattractant wells) and 5 fil of KRP in the inner wells (control wells). The chemoattractants used were the synthetic peptide-formylmethionyl-Ieucyl-phenylalanine (FMLP Sigma-Co St. Louis 23 ), isologous rat serum (IRS). Plates were incubated at 37°C in 95% air/5% CO 2 humidified atmosphere for 150 min. Migrations were measured as the front lead of migration (at least 10 PMN) under the microscope (magnification 24) using a calibrated eye piece i.e. as the distance traversed by the cells from the border of the middle well towards the chemoattractants wells (oriented migration) or the control wells (random migration). Results are expressed in arbitrary units: one unit representing about 360 fim.
B - Classical Boyden chamber techniqu~ modified by Keller et al.1O FMLP, at the concentration of 10 nmolll was used as chemoattractant placed in the lower compartment. For spontaneous migration measurements, we observed the PMN migration without any addition of chemoattractant in the two parts of the chamber. 0.1 ml of cell suspension adjusted to 5 x 106 cells/ml was added to the upper compartment of the chemotactic chamber. Between the two compartments was placed one cellulosic filter (Millipore) of pore diameter 3 fim. The chambers were incubated for 90 min. At the end of the incubation ethanol was added to fix at the cells onto the filter after which they were stained with hematoxylin. Cell migration in five high-power fields for triplicate filters was assessed using the leading front technique 3o • The mean of these values was calculated with s.e.m. and used for statistical analysis using Student's t test.
3rc.
Results
1. Evaluation of the exudative phase and leucocyte migration during various inflammatory reactions Following the injection of various solutions into the rat pleural cavity we have comparatively observed the evolution of the exudates during the first hours of the reaction. Table 1 shows the evolution of the leucocyte content with the mean exudate volumes collected at different times. The volume of the exudates determined by the injection of isologous serum or CaPP does not vary during the first four hours while PBS is gradually subsided. During this time the exudates obtained with the two immune reactions exhibit an enhancement more rapidly reached with the Arthus reaction. Leucocyte amount of the exudates induced by isologous serum or CaPP suspension enhances gradually during the inflammatory processes while that induced by PBS is stable after 1 hour. The Arthus reaction determines an increase in leucocyte content similar to that observed after isologous serum or CaPPo Delayed hypersensitivity reaction induced a weaker and slower increase of cells. The percentages of PMN (Table 2) increase during the various inflammatory reactions. The results obtained indicated that the exudative phase as well as the leucocyte emigration differed according to
132 . M. Roch-Arveiller, A. Perianin, D. Pham Huy and J. P. Giroud Table 1. Evolution of the exudates during inflammatory reaction
1 hour
Substances injected in the pleural cavity N* Isologous serum PBS CaPP Arthus reaction Delayed hypersensitivity (Bordetella pertussis)
13 11
12
3 hours N*
V* 1 0,6 1
73 12 70
4 hours
V' 1 0,2 1
N*
V*
78 10 60 97
1
16 hours N*
V*
29
0,3
1 0,8
* N = Number of cells collected per exudate (x 106 ) * V = Volume of exudate per rat (ml) Table 2. Percentage of mononuclear and polynuclear cells of the pleural exudates Substances injected in the pleural cavity
o hour cells obtained after a washing of the pleural cavity mononuclear PMN PMN cells
Isologous serum P.B.S. CaPP Arthus reaction
10 10 10
90 90 90 90
Delayed hypersensitivity (Bordetella pertussis)
10
90
10
Migratory distances
+
in arbitrary units
•
4
3
2
s. e. m.
o
5
.
40 12 45
••--
isologous serum
PBS CaPP Arthus delayed hypersensit ivity
ii
iii
*... : P < 0.001 Fig. 1. Random migration of pleural PMN (Agarose assay). Each
bar gives the mean tnigratory distam:es of the spontaneous migra-
tion of PMN (7 experiments). A statistically significant difference was designed by (p < 0.001).
1 hour
3 hours
4 hours
mononuclear cells
PMN
mononuclear cells
PMN
60 88 55
93 70
7 30
90 75 90 90
mononuclear cells
10 25 10 10 16 hours 50 50
the inflammatory stimuli. The evolution of the exudates collected after the injection of PBS differs from the other experimental models examined and leads us to distinguish this experimental procedure from the others. In order to analyse the modifications related to the leucocyte emigration we have observed the in vitro migration (chemokinesis and chemotaxis) of PMN harvested in the pleural cavity after the induction of those pleurisies.
2. Study of random migration PMN recruited in the pleural cavity after the injection of CaPP or heat decomplemented isologous serum (DIRS) exhibited comparable spontaneous migration in agarose assay (Fig. 1). After the injection of PBS, the random migration of the PMN is lower but not significantly different from those experimental models . With the Boyden chamber technique (Fig. 2) no significant difference in random migration of cells collected after non immunological reactions was observed. However, the two immune reactions inhibited the cellular random migration. The two models of assessment show a significantly depressed migratory process only for the two immunological reactions.
Modulation of Neutrophil Chemotaxis by Inflammation . 133
120
* ** ***
[J isologous
p< 0.02 p< 0.01 p< 0.001
C
serum
Ii PBS
=
ns
lI
120
CaPP
110
-
E ai 100
iii delayed
vi
+1
E
hype rsensi!i vi! y
ai lOO
vi
E
90
ns
ns
C
1
II II I
IV
70
E
0
*
=
60
... .-en
* .l.
-•-• --•
•
I II 1 I I; I II I II
==
70
E
ii
IE
80
C'l
*
IE Ca PP
**
:4..
c 80 0
...
E
+1
90
~
CII
110
.arthus
isologous serum
60
1
-
arthus
; ; delayed hypersensiti vity
** *
p< * p< ** p < ***
0 .02 0 .01 0 .001
** * .l
-•• -• -• ii ii
Chemotaxis
chemokinesis
Fig. 2. Random migration of pleural PMN (Boyden chamber technique). Evaluation of the mean (7 experiments) migrations performed by PMN recruted after various inflammatory processes without chemotactic gradient.
Fig. 4. PMN migration oriented by FMLP (Boyden chamber technique). Migra!ory distances • s. e. m . in arbitrary units
Migratory distances
•
s . e. m .
in arbitrary units
5
4 ~*
3 2
* *---,---,
r-***,---,
~
I
C
isologous serum
I
PBS
§5
CaPP
•!!
6
5 Arthus delayed hypersensit ivity
~I • :== ••-== ....
7
4
3
~
ii
*** , p < 0.001 Fig. 3. PMN migration oriented by FMLP (Agarose assay). Bars indicated the oriented migration of elicited PMN stimulated by the synthetic peptide FMLP (10- 7 MIl). Each result is the mean of 7 determinations. Statistically significant differences were designed by p < 0.001.
2
1 iii iii ii ii
ii ii ii ii ii
•
ii ii
[]
isologous serum
I
PBS
• iii
CaPP Arthus delayed hypersensitivity
** : p
(0.01
***: P < 0.001
iiii
Fig.S. PMN migration oriented by isologous serum (agarose assay). Bars give the mean oriented migration of elicited PMN stimulated by undiluted isologous rat serum (7 experiments).
134 . M. Roch-Arveiller, A. Perianin, D. Pham Huy and J. P. Giroud
3. Study of PMN chemotaxis induced by FMLP When the synthetic peptide, FMLP, is used as chemoattractant we observed, in agarose assay a similar oriented migration (Fig. 3) for the PMN collected after injection of isologous serum, CaPP crystals or PBS. With the Boyden chamber technique (Fig. 4), the injection of non irritant fluids into the pleural cavity allows a good migration of the PMN through the filter towards FMLP (10- 9 M). However, as we have previously shown 19 chemotactic response of PMN collected after the injection of CaPP is depressed by the inflammatory reaction. With the two techniques of chemotactic assessment we observed an inhibited reactivity of the cells collected after the two immune inflammatory reactions.
4. Study of PMN chemotaxis induced by isologous serum As shown in Fig. 5 rat isologous serum provided a higher chemotactic response than FMLP of PMN recruited in all the pleural exudates examined. The best response was observed (agarose assay) with PMN collected after the administration of isologous serum or CaPP while the immune reactions depressed the chemotactic response of the pleuralleucocytes. PBS determined an intermediate response non significantly different from those obtained in the two other cases. Discussion All these data demonstrated a great variation of PMN chemotactic responsiveness with the cell source and the chemoattractants used. Moreover, some differences can be evidenced by one technique of assessment and not by the others. These observations are sufficient to explain, at least in part, the contradicting effects described in the literature3 and it appears necessary to define precisely all the experimental conditions in which the cells are collected and tested. With agarose assay we can distinguish locomotor reactivity of PMN harvested after immune inflammatory reactions from that of PMN harvested after non immune inflammatory reactions. Chemokinetic and chemotactic responses to various chemoattractants are inhibited in the first case and non significantly affected in the second. With the Boyden chamber technique the inhibition of random or oriented migration of PMN harvested after immune inflammatory reactions is also observed after the injection of an irritant such as calcium pyrophosphate. This technique shbws higher chemotactic response to FMLP than agarose assay and seems more reliable to detect differences between chemotactic responsiveness. The modification of chemotactic reactivity by inflammatory reactions does not seem related to the extent of the
leucocyte migration into the pleural cavity since in four hours. We harvested a similar volume of exudate and a
similar amount of PMN after an Arthus reaction or the injection of isologous serum while PMN exhibit important differences of locomotor reactivity. On the other side the few PMN collected 4 hours after injection of PBS does not exhibit significant variations of reactivity as compared to that of cells harvested 4 hours after injection of isologous serum. Migration in vivo does not seem strickly correlated to the modification of chemotactic or chemokinetic activities assessed in vitro. The difference of chemotactic reactivity can be linked to the nature of cells migrating towards the inflammatory foci. Several subpopulations of PMN exhibiting various functional activities can be collected in the exudates produced during various experimental pleurisies. An other cause of locomotor impairment might be due to an alteration of cellular locomotor apparatus since we always observed an inhibition of random migration in the same time that oriented migration. However, we have none direct evidence of this hypothesis. Otherwise, impaired responsiveness of exudate neutrophils has already been reported. Ward and Becker25 have suggested that this phenomenon might be due to chemotactic deactivation. In some experiments performed with rabbit peritoneal and blood borne neutrophils 9 those two types of cells responded equally well to a gradient of chemotactic exudate fluid. It has been postulated that deactivation may occur non specifically secondary to enhanced neutrophil adhesion to the substratus5 • However, Ackerman et aU have shown that alterations of adhesion and directional migration in vitro were not always predictive of a change in inflammatory cell locomotion in vivo. This raises the question of the correlation between our results obtained in vitro and the events which occur during pathological diseases in vivo. It is therefore clear that a precise understanding of our experimental models provides some approaches to control adverse leucocyte accumulation during chronic inflammatory diseases. Moreover, these experimental processes evidenced the modulation by certain groups of drugs, particularly immunomodulating agents20 of the PMN chemotactic responsiveness according to the physiopathological state of the cells.
References 1 Ackerman N, Martinez S, Thieme T, Mirkovich A (1982) Relationship between adherence, chemotaxis and the accumulation of rat PMN Leukocytes at an inflammatory site. J Pharm EXf Therap 221: 701-707 Boyden S (1962) The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J Exp Med
115:453-466
Clawson CC, White JB, Repine JE (1978) The Chediak Syndrome. Am J Path 92: 745-751 Dieppe PA, Willoughby DA, Huskisson EC, Arrigoni-Martelli E (1976) Pertussis vaccine pleurisy: a model of delayed hypersensitivity. Agents and Actions 6: 618-621 3
Hi~ashi
Modulation of Neutrophil Chemotaxis by Inflammation . 135 5 Fehr ], Dahinden C (1979) Modulating influence of chemotactic factor - induced cell adhesiveness on granulocyte function.] Clin Invest 64: 8-16 6 Gallin JI, Seligmann BE, Fletcher MP (1983) Dynamics of human neutrophil receptors for the chemoattractant f-Met-LeuPhe. In: Keller HU and Till GO (Eds) Leukocyte Locomotion and Chemotaxis. Agents and Actions, Suppl Vol 12, p: 290-308 7 Giroud JP, Roch-Arveiller M, Muntaner 0 (1978) Prelevement repete des polynucleaires dans la cavite pleurale du rat. Application a I'etude du chimiotactisme. Nelle Rev Fse Hemat 20: 535-543 8 Keller HU (1982) Shape, motility and locomotor responses of neutrophil granulocytes. Agents and Actions 12 (Supp AAS 12): 54-72 9 Keller HU, Cottier H (1984) Comparison of locomotion, chemotaxis and adhesiveness of rabbit neutrophils from blood and peritoneal exudates. Blood Cells 10: 45-60 10 Keller HU, Gerber H, Hess MW, Cottier H (1976) Studies on the regulation of the neutrophil chemotactic response using a rapid reliable method for measuring random migration and chemotaxis of neutrophil granulocytes. Agents and Actions 6: 326-339 11 Keller HU, Wilkinson PC, Abercrombie M, Becker EL, Hirsch JG, Miller ME, Ramsey WS, Zigmond S (1977) A proposal for the definition of terms related to locomotion of leucocytes and other cells. Clin and Exp Immunology 27: 377-380 12 Mandell LA (1982) Modulators of host defence in malignancy. In: Easmon CSF and Gaya HA (Eds) Proceedings of 2nd intern. Symposium on Infections in Immunocompromised Hosts, pp 37. Academic press, London 13 Miller ME, Norman ME, Koblenzer RJ, Schonauer T (1973) A new familial defect of neutrophil movement. J lab clin med 82: 1-8 14 Miller ME, Oski FA, Harris MB (1971) Lazy leukocytes syndrome - A new disorders of neutrophil function. Lancet 1: 665-669 15 Nelson RD, Quie RG, Simmons RL (1975) Chemotaxis under agarose: A new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J Immunol 115: 1650-1656 16 Perianin A, Labro MT, Hakim J (1982) Chemokinetic activity of N-formyl-methionyl-Ieucyl-phenylalanine on human neut-
rophils and its modulation by phenylbutazone. Biochem Pharmacol31: 3071-3076 17 Quie PG (1983) Clinical disorders of phagocyte locomotion. In: Keller HU and Till GO (Eds) Leukocyte Locomotion and Chemotaxis, pp 398-415, Birkiiuser Verlag, Agents and Actions, SUff112: Quie PG, Cates KL (1978) Clinical manifestations of disorders of neutrophil. In: Gallin JI and Quie PG (Eds) Leukocyte Chemotaxis, pp 307-328. Raven Press, New York 19 Roch-Arveiller M, Abita JP, Giroud JP (1980) Modification du chimiotactisme des polynucleaires de rat au cours de l'inflammation experimentale. C R Ac Sc 290: 1545-1548 20 Roch-Arveiller M, Tissot M, Moachon L, Giroud JP (1982) Effect of immunomodulating agents on leucocyte chemotaxis and cyclic nucleotides. Agents and Actions 12: 353-359 21 Schiffmann E (1981) Molecular events in leukocyte chemotaxis: their possible roles in processing the chemical signal. Biosciences reports 1: 89-99 22 Schiffmann E (1982) Leukocyte chemotaxis. Ann Rev Physiol 44: 553-568 23 Schiffmann E, Corcoran BA, Wahl SM (1975) N-formyl propionyl peptides as chemoattractants for leucocytes. Proc Nat Acad Sci (USA) 72: 1059-1062 24 Snyderman R, Goetzl EJ (1981) Molecular and cellular mechanisms of leucocyte chemotaxis. Science 213: 830-837 25 Ward PA, Becker EL (1968) The deactivation of rabbit neutrophils by chemotactic factor and the nature of the deactivatable esterase. J exp med 127: 699-710 26 Wilkinson PC (1982) Chemotaxis and Inflammation (2nd edition), pp 1-26 Churchill Livingston, Edinburgh 27 Wilkinson PC, Allan RB (1978) Assay systems for measuring leucocyte locomotion. An overview. In: Gallin JI and Quie P (Eds) Leukocyte Chemotaxis, pp 1-24. Raven press, New York 28 Willoughby DA, Dunn CJ, Yamamoto S, Capasso F, Deporter DA and Giroud JP (1975) Calcium pyrophosphate - induced pleurisy in rat: a new model of acute inflammation. Agents and Actions 5: 35-38 29 Yamamoto S, Dunn DJ, Deporter DA, Capasso F, Willoughby DA, Huskisson EC (1975) A model for the quantitative study of Arthus (Immunologic) hypersensitivity in rats. Agents and Actions 5: 374-377 30 Zigmond SH, Hirsch JG (1973) Leukocyte locomotion and chemotaxis. New methods for evaluation and demonstration of a cell derived chemotactic factor. J exp med 137: 387-410
Received April 16, 1984 . Accepted July 13, 1984
Key words: Chemotaxis - Neutrophils - Migration - Inflammatory reactions Professeur J. P. Giroud, Departement de Pharmacologie, Hopital Cochin, 27, rue du Fbg. St. Jacques, F 75674 Paris Cedex 14, France
Modification of Neutrophil Chemotactic Responsiveness During Various Inflammatory Reactions M. Roch-Arveiller, A. Perianin, D. Pham Huy and J. P. Giroud (ERA CNRS 629), Department of Pharmacology, 27 rue du Fbg. St-Jacques75014 Paris
SUMMARY Various inflammatory reactions have been induced in order to examine the chemotactic response of polymorphonuclear leucocytes (PMN) collected under various experimental conditions. Cells were harvested from the pleural cavity of rats after the induction of three acute non specific inflammatory reactions and two immune reactions. The results obtained with two techniques of chemotactic assessment (agarose assay and Boyden chamber technique) demonstrated a variation of chemotactic response depending on the cell source and the chemoattractants used. Using agarose assay, we distinguished locomotor reactivity of PMN harvested after immune inflammatory reactions from that of PMN harvested after non immune inflammatory reactions. Chemokinetic and chemotactic responses to various chemoattractants were inhibited in the first case and not affected in the second. Using the Boyden chamber technique, inhibition of random or oriented migration of PMN harvested after immune inflammatory reactions after the injection of a non antigenic irritant such as calcium pyrophosphate crystals (CaPP) was also observed.
Introduction The rapid accumulation of phagocytic wandering cells at sites of inflammation, infection or antigenic penetration appears to be essential for the defence of the host against various injuries. One mechanism which can lead to the local accumulation of motile cells is chemotaxis i.e. the unidirectional migration of cells along a gradient of chemoattractant(s). This parameter must be distinguished from chemokinesis in which sfeed of the cells is modified without change of directions,l , 26, 27. The directed migration of leucocytes in response to chemotactic gradients is a complex series of biochemical This paper is based on a speech held at the IXth European Congress
of Pathology in Hamburg 1983 in the session C 3 "Biochemistry of Inflammation" . 0344-0338/85/0180-0130$3.50/0
phenomena requiring recognition of chemical signals by the cell membrane, the triggering of energy-forming processes within the cell and the translation of this energy by cytostructural elements into movement. The events which result in the directed migration of motile cells as a consequence of their exposure to chemotactic gradients are still unknown. However several lines of evidence suggest that chemotactic factor binding to leucocyte, divalent cation fluxes activation of certain metabolic pathways and polymerisation of actomyosin-like molecules within the cells are required for cell movemenrl,22. Directed migration appears to result from the adaptation of the cells to local changes in stimulus concentration 6, 24. An intact phagocytic system is necessary for normal host defence and a rapid migration into tissues where
surface defences have been breached, is a neutrophil function essential for a good health. These experimental obser© 1985 by Gustav Fischer Verlag, Stuttgart
Modulation of Neutrophil Chemotaxis by Inflammation . 131
vations have been supported recently by several clinical observations which suggest an association between susceptibility to infection and depressed neutrophil chemotactic responsiveness in patients with recurrent severe infections. Numerous reports of intrinsic cellular defects of locomotion in individual patients with serious infections disease problems suggest that this may be a frequent basis for diminished host defence 13 , 17, 18. Defective phagocytic cell locomotion in patients may be the consequence of a variety of causes: - intrinsic cellular defects of locomotion (lazy leucocyte syndrome of Miller 14 and many other syndromes susceptible to exhibit recurrent infections). This defect may be transient in certain patients. - defective generation of chemotactic factors. - inhibition of chemotactic factors described by Mandelll2 in patients with malignancies. The multiple examples of clinical locomotory disorders led us to examine the cellular behaviour of polymorphonuclear leucocytes (PMN) harvested during various experimental inflammatory reactions. Previous studies 19 have demonstrated an inhibition of PMN chemotactic responsiveness during an acute non specific inflammatory reaction (pleurisy after injection of a suspension of CaPP crystals). We want to present here the modification of neutrophil chemotactic response observed after the induction of various inflammatory reactions of non immune and immune origin. Material and Methods
1. Cell Collection Pathogen free, male Sprague-Dawley rats (180-200 g) were used for all experiments. Leucocytes were collected from the pleural cavity 4 hours after the injection of - 1 ml of isologous serum heated fordecomplementation 7, - 1 ml of Phosphate Buffer Saline (PBS), - 1 ml of a suspension of Calcium pyrophosphate crystals (CaPP) (1 %) in saline28 • On the other hand, we have harvested rat PMN after the induction of two immune inflammatory reactions: - a reversed passive Arthus reaction in the pleural cavity29, - a reaction of delayed hypersensitivity on rats sensitized to Bordetella pertussis 4 • The cells were washed twice and resuspended in appropriate medium: Krebs' solution for agarose assays, Hanks' solution with bovine serum albumine (1%) for Boyden chamber technique.
2. Chemotactic assessment It has been performed by two techniques:
A - Migration under agarose
Spontaneous and oriented migration were measured by the modified version of the agarose method15, 16 4 ml of 0,75% (v/v) indubiose A-37 (Industrie biologique fran<;aise - France) in Krebs Ringer Phosphate (KRP) pH 6.8 containing 10% of heat-inactivated foetal calf serum, was poured into small tissue culture Petri
dishes (Falcon 15 X 55 mm - Lab. Express Servo France). Four sets of 3 wells, 2.5 mm in diameter and spaced 2.5 mm apart were cut out using a template; 5 fil of leucocyte suspension (i.e. 5 x 105 PMN) was placed in each of the middle wells (PMN well), 5 fil of chemotactic factors in the outer wells (chemoattractant wells) and 5 fil of KRP in the inner wells (control wells). The chemoattractants used were the synthetic peptide-formylmethionyl-Ieucyl-phenylalanine (FMLP Sigma-Co St. Louis 23 ), isologous rat serum (IRS). Plates were incubated at 37°C in 95% air/5% CO 2 humidified atmosphere for 150 min. Migrations were measured as the front lead of migration (at least 10 PMN) under the microscope (magnification 24) using a calibrated eye piece i.e. as the distance traversed by the cells from the border of the middle well towards the chemoattractants wells (oriented migration) or the control wells (random migration). Results are expressed in arbitrary units: one unit representing about 360 fim.
B - Classical Boyden chamber techniqu~ modified by Keller et al.1O FMLP, at the concentration of 10 nmolll was used as chemoattractant placed in the lower compartment. For spontaneous migration measurements, we observed the PMN migration without any addition of chemoattractant in the two parts of the chamber. 0.1 ml of cell suspension adjusted to 5 x 106 cells/ml was added to the upper compartment of the chemotactic chamber. Between the two compartments was placed one cellulosic filter (Millipore) of pore diameter 3 fim. The chambers were incubated for 90 min. At the end of the incubation ethanol was added to fix at the cells onto the filter after which they were stained with hematoxylin. Cell migration in five high-power fields for triplicate filters was assessed using the leading front technique 3o • The mean of these values was calculated with s.e.m. and used for statistical analysis using Student's t test.
3rc.
Results
1. Evaluation of the exudative phase and leucocyte migration during various inflammatory reactions Following the injection of various solutions into the rat pleural cavity we have comparatively observed the evolution of the exudates during the first hours of the reaction. Table 1 shows the evolution of the leucocyte content with the mean exudate volumes collected at different times. The volume of the exudates determined by the injection of isologous serum or CaPP does not vary during the first four hours while PBS is gradually subsided. During this time the exudates obtained with the two immune reactions exhibit an enhancement more rapidly reached with the Arthus reaction. Leucocyte amount of the exudates induced by isologous serum or CaPP suspension enhances gradually during the inflammatory processes while that induced by PBS is stable after 1 hour. The Arthus reaction determines an increase in leucocyte content similar to that observed after isologous serum or CaPPo Delayed hypersensitivity reaction induced a weaker and slower increase of cells. The percentages of PMN (Table 2) increase during the various inflammatory reactions. The results obtained indicated that the exudative phase as well as the leucocyte emigration differed according to
132 . M. Roch-Arveiller, A. Perianin, D. Pham Huy and J. P. Giroud Table 1. Evolution of the exudates during inflammatory reaction
1 hour
Substances injected in the pleural cavity N* Isologous serum PBS CaPP Arthus reaction Delayed hypersensitivity (Bordetella pertussis)
13 11
12
3 hours N*
V* 1 0,6 1
73 12 70
4 hours
V' 1 0,2 1
N*
V*
78 10 60 97
1
16 hours N*
V*
29
0,3
1 0,8
* N = Number of cells collected per exudate (x 106 ) * V = Volume of exudate per rat (ml) Table 2. Percentage of mononuclear and polynuclear cells of the pleural exudates Substances injected in the pleural cavity
o hour cells obtained after a washing of the pleural cavity mononuclear PMN PMN cells
Isologous serum P.B.S. CaPP Arthus reaction
10 10 10
90 90 90 90
Delayed hypersensitivity (Bordetella pertussis)
10
90
10
Migratory distances
+
in arbitrary units
•
4
3
2
s. e. m.
o
5
.
40 12 45
••--
isologous serum
PBS CaPP Arthus delayed hypersensit ivity
ii
iii
*... : P < 0.001 Fig. 1. Random migration of pleural PMN (Agarose assay). Each
bar gives the mean tnigratory distam:es of the spontaneous migra-
tion of PMN (7 experiments). A statistically significant difference was designed by (p < 0.001).
1 hour
3 hours
4 hours
mononuclear cells
PMN
mononuclear cells
PMN
60 88 55
93 70
7 30
90 75 90 90
mononuclear cells
10 25 10 10 16 hours 50 50
the inflammatory stimuli. The evolution of the exudates collected after the injection of PBS differs from the other experimental models examined and leads us to distinguish this experimental procedure from the others. In order to analyse the modifications related to the leucocyte emigration we have observed the in vitro migration (chemokinesis and chemotaxis) of PMN harvested in the pleural cavity after the induction of those pleurisies.
2. Study of random migration PMN recruited in the pleural cavity after the injection of CaPP or heat decomplemented isologous serum (DIRS) exhibited comparable spontaneous migration in agarose assay (Fig. 1). After the injection of PBS, the random migration of the PMN is lower but not significantly different from those experimental models . With the Boyden chamber technique (Fig. 2) no significant difference in random migration of cells collected after non immunological reactions was observed. However, the two immune reactions inhibited the cellular random migration. The two models of assessment show a significantly depressed migratory process only for the two immunological reactions.
Modulation of Neutrophil Chemotaxis by Inflammation . 133
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Fig. 2. Random migration of pleural PMN (Boyden chamber technique). Evaluation of the mean (7 experiments) migrations performed by PMN recruted after various inflammatory processes without chemotactic gradient.
Fig. 4. PMN migration oriented by FMLP (Boyden chamber technique). Migra!ory distances • s. e. m . in arbitrary units
Migratory distances
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134 . M. Roch-Arveiller, A. Perianin, D. Pham Huy and J. P. Giroud
3. Study of PMN chemotaxis induced by FMLP When the synthetic peptide, FMLP, is used as chemoattractant we observed, in agarose assay a similar oriented migration (Fig. 3) for the PMN collected after injection of isologous serum, CaPP crystals or PBS. With the Boyden chamber technique (Fig. 4), the injection of non irritant fluids into the pleural cavity allows a good migration of the PMN through the filter towards FMLP (10- 9 M). However, as we have previously shown 19 chemotactic response of PMN collected after the injection of CaPP is depressed by the inflammatory reaction. With the two techniques of chemotactic assessment we observed an inhibited reactivity of the cells collected after the two immune inflammatory reactions.
4. Study of PMN chemotaxis induced by isologous serum As shown in Fig. 5 rat isologous serum provided a higher chemotactic response than FMLP of PMN recruited in all the pleural exudates examined. The best response was observed (agarose assay) with PMN collected after the administration of isologous serum or CaPP while the immune reactions depressed the chemotactic response of the pleuralleucocytes. PBS determined an intermediate response non significantly different from those obtained in the two other cases. Discussion All these data demonstrated a great variation of PMN chemotactic responsiveness with the cell source and the chemoattractants used. Moreover, some differences can be evidenced by one technique of assessment and not by the others. These observations are sufficient to explain, at least in part, the contradicting effects described in the literature3 and it appears necessary to define precisely all the experimental conditions in which the cells are collected and tested. With agarose assay we can distinguish locomotor reactivity of PMN harvested after immune inflammatory reactions from that of PMN harvested after non immune inflammatory reactions. Chemokinetic and chemotactic responses to various chemoattractants are inhibited in the first case and non significantly affected in the second. With the Boyden chamber technique the inhibition of random or oriented migration of PMN harvested after immune inflammatory reactions is also observed after the injection of an irritant such as calcium pyrophosphate. This technique shbws higher chemotactic response to FMLP than agarose assay and seems more reliable to detect differences between chemotactic responsiveness. The modification of chemotactic reactivity by inflammatory reactions does not seem related to the extent of the
leucocyte migration into the pleural cavity since in four hours. We harvested a similar volume of exudate and a
similar amount of PMN after an Arthus reaction or the injection of isologous serum while PMN exhibit important differences of locomotor reactivity. On the other side the few PMN collected 4 hours after injection of PBS does not exhibit significant variations of reactivity as compared to that of cells harvested 4 hours after injection of isologous serum. Migration in vivo does not seem strickly correlated to the modification of chemotactic or chemokinetic activities assessed in vitro. The difference of chemotactic reactivity can be linked to the nature of cells migrating towards the inflammatory foci. Several subpopulations of PMN exhibiting various functional activities can be collected in the exudates produced during various experimental pleurisies. An other cause of locomotor impairment might be due to an alteration of cellular locomotor apparatus since we always observed an inhibition of random migration in the same time that oriented migration. However, we have none direct evidence of this hypothesis. Otherwise, impaired responsiveness of exudate neutrophils has already been reported. Ward and Becker25 have suggested that this phenomenon might be due to chemotactic deactivation. In some experiments performed with rabbit peritoneal and blood borne neutrophils 9 those two types of cells responded equally well to a gradient of chemotactic exudate fluid. It has been postulated that deactivation may occur non specifically secondary to enhanced neutrophil adhesion to the substratus5 • However, Ackerman et aU have shown that alterations of adhesion and directional migration in vitro were not always predictive of a change in inflammatory cell locomotion in vivo. This raises the question of the correlation between our results obtained in vitro and the events which occur during pathological diseases in vivo. It is therefore clear that a precise understanding of our experimental models provides some approaches to control adverse leucocyte accumulation during chronic inflammatory diseases. Moreover, these experimental processes evidenced the modulation by certain groups of drugs, particularly immunomodulating agents20 of the PMN chemotactic responsiveness according to the physiopathological state of the cells.
References 1 Ackerman N, Martinez S, Thieme T, Mirkovich A (1982) Relationship between adherence, chemotaxis and the accumulation of rat PMN Leukocytes at an inflammatory site. J Pharm EXf Therap 221: 701-707 Boyden S (1962) The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J Exp Med
115:453-466
Clawson CC, White JB, Repine JE (1978) The Chediak Syndrome. Am J Path 92: 745-751 Dieppe PA, Willoughby DA, Huskisson EC, Arrigoni-Martelli E (1976) Pertussis vaccine pleurisy: a model of delayed hypersensitivity. Agents and Actions 6: 618-621 3
Hi~ashi
Modulation of Neutrophil Chemotaxis by Inflammation . 135 5 Fehr ], Dahinden C (1979) Modulating influence of chemotactic factor - induced cell adhesiveness on granulocyte function.] Clin Invest 64: 8-16 6 Gallin JI, Seligmann BE, Fletcher MP (1983) Dynamics of human neutrophil receptors for the chemoattractant f-Met-LeuPhe. In: Keller HU and Till GO (Eds) Leukocyte Locomotion and Chemotaxis. Agents and Actions, Suppl Vol 12, p: 290-308 7 Giroud JP, Roch-Arveiller M, Muntaner 0 (1978) Prelevement repete des polynucleaires dans la cavite pleurale du rat. Application a I'etude du chimiotactisme. Nelle Rev Fse Hemat 20: 535-543 8 Keller HU (1982) Shape, motility and locomotor responses of neutrophil granulocytes. Agents and Actions 12 (Supp AAS 12): 54-72 9 Keller HU, Cottier H (1984) Comparison of locomotion, chemotaxis and adhesiveness of rabbit neutrophils from blood and peritoneal exudates. Blood Cells 10: 45-60 10 Keller HU, Gerber H, Hess MW, Cottier H (1976) Studies on the regulation of the neutrophil chemotactic response using a rapid reliable method for measuring random migration and chemotaxis of neutrophil granulocytes. Agents and Actions 6: 326-339 11 Keller HU, Wilkinson PC, Abercrombie M, Becker EL, Hirsch JG, Miller ME, Ramsey WS, Zigmond S (1977) A proposal for the definition of terms related to locomotion of leucocytes and other cells. Clin and Exp Immunology 27: 377-380 12 Mandell LA (1982) Modulators of host defence in malignancy. In: Easmon CSF and Gaya HA (Eds) Proceedings of 2nd intern. Symposium on Infections in Immunocompromised Hosts, pp 37. Academic press, London 13 Miller ME, Norman ME, Koblenzer RJ, Schonauer T (1973) A new familial defect of neutrophil movement. J lab clin med 82: 1-8 14 Miller ME, Oski FA, Harris MB (1971) Lazy leukocytes syndrome - A new disorders of neutrophil function. Lancet 1: 665-669 15 Nelson RD, Quie RG, Simmons RL (1975) Chemotaxis under agarose: A new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J Immunol 115: 1650-1656 16 Perianin A, Labro MT, Hakim J (1982) Chemokinetic activity of N-formyl-methionyl-Ieucyl-phenylalanine on human neut-
rophils and its modulation by phenylbutazone. Biochem Pharmacol31: 3071-3076 17 Quie PG (1983) Clinical disorders of phagocyte locomotion. In: Keller HU and Till GO (Eds) Leukocyte Locomotion and Chemotaxis, pp 398-415, Birkiiuser Verlag, Agents and Actions, SUff112: Quie PG, Cates KL (1978) Clinical manifestations of disorders of neutrophil. In: Gallin JI and Quie PG (Eds) Leukocyte Chemotaxis, pp 307-328. Raven Press, New York 19 Roch-Arveiller M, Abita JP, Giroud JP (1980) Modification du chimiotactisme des polynucleaires de rat au cours de l'inflammation experimentale. C R Ac Sc 290: 1545-1548 20 Roch-Arveiller M, Tissot M, Moachon L, Giroud JP (1982) Effect of immunomodulating agents on leucocyte chemotaxis and cyclic nucleotides. Agents and Actions 12: 353-359 21 Schiffmann E (1981) Molecular events in leukocyte chemotaxis: their possible roles in processing the chemical signal. Biosciences reports 1: 89-99 22 Schiffmann E (1982) Leukocyte chemotaxis. Ann Rev Physiol 44: 553-568 23 Schiffmann E, Corcoran BA, Wahl SM (1975) N-formyl propionyl peptides as chemoattractants for leucocytes. Proc Nat Acad Sci (USA) 72: 1059-1062 24 Snyderman R, Goetzl EJ (1981) Molecular and cellular mechanisms of leucocyte chemotaxis. Science 213: 830-837 25 Ward PA, Becker EL (1968) The deactivation of rabbit neutrophils by chemotactic factor and the nature of the deactivatable esterase. J exp med 127: 699-710 26 Wilkinson PC (1982) Chemotaxis and Inflammation (2nd edition), pp 1-26 Churchill Livingston, Edinburgh 27 Wilkinson PC, Allan RB (1978) Assay systems for measuring leucocyte locomotion. An overview. In: Gallin JI and Quie P (Eds) Leukocyte Chemotaxis, pp 1-24. Raven press, New York 28 Willoughby DA, Dunn CJ, Yamamoto S, Capasso F, Deporter DA and Giroud JP (1975) Calcium pyrophosphate - induced pleurisy in rat: a new model of acute inflammation. Agents and Actions 5: 35-38 29 Yamamoto S, Dunn DJ, Deporter DA, Capasso F, Willoughby DA, Huskisson EC (1975) A model for the quantitative study of Arthus (Immunologic) hypersensitivity in rats. Agents and Actions 5: 374-377 30 Zigmond SH, Hirsch JG (1973) Leukocyte locomotion and chemotaxis. New methods for evaluation and demonstration of a cell derived chemotactic factor. J exp med 137: 387-410
Received April 16, 1984 . Accepted July 13, 1984
Key words: Chemotaxis - Neutrophils - Migration - Inflammatory reactions Professeur J. P. Giroud, Departement de Pharmacologie, Hopital Cochin, 27, rue du Fbg. St. Jacques, F 75674 Paris Cedex 14, France