Int. Archs Allergy appl. Immun. 53 : 29-36 (1977)

Different Concentrations of Chemotactic Factors Can Produce Attraction or Migration Inhibition of Leukocytes T. W.Jungi Schweizerisches Forschungsinstitut, Medizinische Abteilung, Davos

Abstract. Parallel tests were conducted utilizing, the capillary tube migration test and the Boyden chamber assay, in order to determine whether the decrease in leukocyte chemotaxis that occurs if overoptimal cytotaxin concentrations are applied is due to migration inhibition. Overoptimal doses of casein produced decreased chemotactic response and migration in­ hibition for both rabbit macrophages and neutrophils. However, guinea pig neutrophils exhibited no decrease in chemotaxis despite high casein doses. Overoptimal doses of aciddenatured anaphylatoxin produced a decreased chemotactic response and migration inhibition of neutrophils. In both assays, this agent showed no effect upon macrophages. It is concluded that a chemotactic signal at different concentrations can elicit unidirectional migration or migration inhibition. Accordingly, chemotactic leukocyte attraction could be antagonistically regulated not only by serum-derived and lymphocyte-derived migration inhibitory factors but also by high doses of the chemotactic factor itself. Thus, the Boyden chamber technique can measure both chemotactic migration and migration inhibition phenomena.

Chemotaxis and migration inhibition of macrophages are regarded as important mechanisms of cell mediated immunity. In this type of immune response, T cells re­ lease chemotactic and migration inhibitory factors which attract and then trap the cells in the inflammatory focus. It has been pro­ posed [24] that both phenomena, chemotax­ is and migration inhibition, are due to sepa­ rate molecular entities. It is conceivable, Received: May 21, 1975.

however, that the same agent could act as a chemoattractant at some distance from the focus and in higher concentration, at the center of the focus, act as a trapping (migra­ tion inhibitory) substance. Numerous chemotactic factors have been shown in vitro to produce decreased chemo­ taxis when applied in overoptimal concen­ trations [21, 27]. This report is an attempt to clarify whether this phenomenon repre­ sents an artifact of the method or whether it is due to a specific effect of the attractant Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 10:49:29 AM

Introduction

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upon the cells. Such a cellular effect could be expressed as inability of the cells to rec­ ognize the stimulus if presented in too high a concentration or in a response of the rec­ ognizing cells other than unidirectional mi­ gration such as migration inhibition. There­ fore, chemotactic agents have been tested over a broad concentration range in parallel in the Boyden chamber assay [6, 13], and the classical migration inhibition capillary tube test [8, 11]. Casein as both a monocyte and neutrophil cytotaxin and acid-dena­ tured purified anaphylatoxin [28] attracting neutrophils but not macrophages served as chemotactic agents.

Materials and Methods Cells Peritoneal neutrophils were harvested 16 h af­ ter injection of 40 ml of 3.5% w/v sodium casein­ ate solution from rabbits of both sexes (2-3.5 kg). Rabbit peritoneal macrophages were harvested 4 days after injection of 40 ml of paraffin oil (Merck, DAB 7). The cells were washed appropri­ ately and suspended in medium in a concentration of 30X106 test cells per milliliter. One moiety was used for the migration inhibition assay, the other was further diluted to a concentration of 2X 106 test cells per milliliter and then used for the chemotaxis test. In a few experiments, guinea pig exu­ date cells were used for comparison. Peritoneal neutrophils were harvested 4 h after injection of 10 ml of sodium caseinate solution from normal outbred guinea pigs of both sexes (400-800 g), macrophages were collected 4 days after i.p. injec­ tion of 10 ml of paraffin oil. Chemoattractants Casein (alkali-soluble, Bender & Hobein, Zürich), was prepared as stock solutions of 5% w/v and stored at -20 °C in small aliquots. Hog anaphylatoxin prepared according to Wissler [26], was acid-denatured by treatment with acetic acid (pH 4.7), for a prolonged period of time. Such a

preparation, earlier shown to be a polymorphonu­ clear-specific attractant [28], was kindly given to us by Dr. Wissler. Cruder AT preparations with the same properties were used for comparison. Chemotaxis Measurement Chemotaxis was measured using the reversible chemotaxis chamber recently described [13]. This chamber overcomes cell detachment from the fil­ ter after migration, thus eliminating the possibility that a ‘fast’ response provoked by a strong stimu­ lus is not detected because the cells have already detached from the filter when the incubation is terminated. Such a reversing has been shown to be an appropriate maneuver. However, since dislodg­ ing of rabbit neutrophils has earlier been shown to be minimal [13], chambers needed not to be re­ versed in rabbit neutrophil experiments. Unless otherwise stated, incubation times, pH values, fil­ ter processing and other details of methodology were as described previously [13]. Capillary Tube Test Capillary tubes were filled with cells at a con­ centration of 30 X 106/ml. After sealing with semisoft paraffin, they were centrifuged at 200 g. Then, the capillary tubes were cut at the cell-fluid inter­ phase and put into migration inhibition chambers (Minilab, Ouvernay, Canada). Dilutions made with the appropriate medium from the same stock solutions as used in the chemotaxis assay were prepared and filled carefully into the chambers prior to closing them with a coverslip and incubat­ ing at 37 °C. After 6 h (neutrophils) or 24 and 48 h (macrophages), the chambers were placed un­ der an inversion microscope and photographed by a Polaroid camera using a 4-fold magnification objective. The area of cell emigration from the capillary tube was determined planimetrically on the photographs. Migration inhibition was ex­ pressed as percent inhibition with regard to a mi­ gration area from capillary tubes filled with medi­ um only. Migration inhibition of more than 20% was regarded as significant [18]. Media For the capillary tube test, normally Medium 199 (Flow Laboratories, Irvine, Scotland, cat. No. 1-044D) containing 5% v/v fetal calf serum (Flow Laboratories, Irvine, Scotland, cat. No. 4-

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Leukocyte Chemotaxis and Migration Inhibition

Casein concentration. °/0w/w

Casein concentration,“/« w/w

Fig. 2. No decrease of chemotaxis of guinea pig neutrophils when high doses of casein were applied to the lower compartment of a modified Boydcn cham­ ber. 3 experiments are recorded (O, • , □), the solid line representing the arithmetic mean. In all cases, casein was made up as a 5% stock solution.

Fig. 1. Correlation of migration inhibition with decreased chcmotaxis when high doses of casein were applied to rabbit peritoneal neutrophils. Chemotaxis was measured by means of a modified Boydcn chamber [13], casein being added to the lower com­ partment. All 5 experiments recorded (O, A, □, ■, • ) showed similar dose-response patterns, the solid line being the arithmetic mean. Migration inhibition was measured by using the classical capillary tube migra­ tion inhibition assay (casein was added to the chamber fluid). Bars represent means of migration inhibition with regard to casein-free chambers. Casein was made up as a 5% stock solution for both assays.

015E; M 199-FCS) was used; for the chemotaxis experiments, Gey’s medium with antibiotics and containing 2°/o w/v human serum albumin (Gey’s HSA) [27]. Use of M 199 - FCS in the chemotax­ is test seemed to inhibit chemotactic migration sig­ nificantly, although the response pattern was es­ sentially comparable with both media. According­ ly, cells suspended in Gey’s - HSA emigrated to a lesser extent from the capillary tubes although the results obtained with both media did not differ basically. Therefore, in the two assays, preference was given to the medium giving the highest re­ sponse.

Casein as a Chemotactic and Migration Inhibitory Agent For neutrophils. Casein was tested at various concentrations in parallel experi­ ments for leukocyte chemotaxis in the modi­ fied Bovden chamber and for migration in­ hibition in the capillary-tube test. Figure 1 shows the results for rabbit neutrophils, us­ ing a 5% casein stock solution for all dilu­ tions made up with the appropriate medium. Usually, maximal chemotaxis was observed at a concentration of 0.32% casein. More concentrated or more diluted solutions show­ ed decreased chemotaxis. Capillary migra­ tion inhibition was also observed, and this inhibition correlated with the chemotaxis inhibition induced with overoptimal concen­ trations of casein. It is noteworthy, that guinea pig neutro­ phils did not behave as rabbit neutrophils in that higher concentrated casein solutions did not lead to a decreased chemotactic re­ sponse but rather reached a plateau (fig. 2). The restricted solubility of casein did not al­ low testing still higher concentrations which might have been needed for demonstrating migration inhibition in this cell type.

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Results

Fig. 3. Correlation of migration inhibition with decreased chemotaxis when high doses of casein were applied to rabbit peritoneal macrophages. For details, see figure 1. Chemotaxis: means and standard de­ viations (vertical lines) of one typical experiment are recorded. Migration inhibition: percent inhibition of the same experiment with reference to casein-free chambers are illustrated.

For macrophages. Similar results as for rabbit neutrophils were obtained with rabbit and with guinea pig macrophages, although with the latter cell type, a somewhat higher variability was observed in the chemotaxis assay. One typical experiment is illustrated in figure 3. It shows that decreased chemo­ taxis occurred with overoptimal casein con­ centrations which also produced migration inhibition in the capillary-tube assay. Casein-Induced Migration Inhibition Is not Due to Cytotoxic Effects Having established that casein in high concentrations inhibits chemotaxis as well as capillary tube migration, another set of

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experiments was performed to exclude a noxious effect of casein upon cells. Several lines of evidence indicate that true migra­ tion inhibition was measured. Trypan-blue exclusion tests showed that both rabbit neu­ trophils and macrophages were still viable after incubation in overoptimal casein con­ centrations for the same time period as used in the chemotaxis tests as incubation time (3 and 5 h, respectively). Moreover, the casein solutions did not impair leukocyte function as expressed by measurement of chemotactic response towards an optimal stimulus. Neutrophils were incubated for 3*/2 h in con­ centrated casein solutions sufficient to de­ crease chemotaxis, then washed, suspended in casein-free medium for 12 h at 2 °C (to exclude possible tachyphylactic effects) and finally tested for chemotactic responsiveness. Such cells responded normally to casein as an attractant in a Boyden chamber experi­ ment (table I). Correlation of Cell Specificity in Chemo­ taxis and Migration Inhibition Assays Since inhibition was manifested under comparable conditions in two different as­ says, one measuring directed migration, the other inhibition of random migration, it was of interest to verify whether cell specificity of a chemotactic agent is maintained when migration inhibition instead of chemotactic migration is measured. For this purpose, a neutrophil specific cytotaxin, acid-dena­ tured purified anaphylatoxin was used. Varying concentrations were added to the lower compartment of chemotaxis chambers and to the migration inhibition chambers. The results are presented in table II. The data demonstrate that neutrophils recognize and are chemotactically attracted by AT at low concentration (1 0 -7 m ), but chemotactic

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Leukocyte Chemotaxis and Migration Inhibition

Casein concentration Control 3 'A h pretreatment in the lower compart- (no casein) with casein ment used in the 5% 1% 0.5% chemotaxis experi­ ment, % 49 »

5 1 0.5

170 184

34 167 183

72 125 169

14 119 172

a Cells per field; mean of duplicate filters of a typical experiment, 4 fields counted per filter.

attraction is inhibited at higher concentra­ tions. This dose-dependent inhibition is cor­ related with the inhibition observed in the capillary tube assay. In contrast to the re­ sults with neutrophils, macrophages neither responded chemotactically to acid-dena­ tured anaphylatoxin at any dose nor was their capillary tube migration inhibited. This demonstrates again that migration inhi­ bition is one way in which the interaction of leukocytes with a chemotactic agent, in overoptimal concentration, expresses itself. It further suggests that cell specificity of rec­ ognition is maintained when migration inhi­ bition instead of chemotactic migration is the response. Similar results were obtained if crude AT preparations, instead of highly purified AT, were used as stimuli.

Discussion

Previous workers have reported that an overdose of a chemotactic factor leads to a

Table n . Parallel decrease of chemotactic response and migration inhibition in rabbit neutrophils. (Acid dena­ tured anaphylatoxin was used as neutrophil-specific attractant). Moreover, the results demonstrate cell specificity in that rabbit macrophages unlike rabbit neutrophils showed neither a chemotactic nor a mi­ gration inhibition response. Concen­ tration of acid-de­ natured anaphyla­ toxin x 10-7 M 150 50 20 6 3 1

Neutrophils

Macrophages

chemo- migration chemo- migration taxis inhibition taxis inhibition %a



%a

%

15 20 40 70 85 100

100 95 83 66 58 no inhibition

0 0 0 0 0 0

no inhibition no inhibition no inhibition no inhibition no inhibition no inhibition

a 100% = Cells per field obtained when an optimal dose of an attractant was applied to the lower com­ partment. b Migration inhibition is expressed as percent in­ hibition with regard to chambers containing anaphylatoxin-fremedium.

decreased chemotactic response. For exam­ ple, decreased leukotactic activity has been observed for high concentrations of fibrino­ lytic products [21], certain myoglobin preparations [14] and anaphylatoxincocytotaxin-mixtures [27], respectively, whereas all these substances were chemo­ tactic at lower concentrations. This phe­ nomenon has also been described in systems other than mammalian cells. For instance, Escherichia coli is attracted by relatively low concentrations of various sugars and amino acids [1, 2]. Still lower as well as higher concentrations of these agents do not attract the bacteria [2]. However, if such a phenomenon is observed in any system, it

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Table I. Demonstration of unimpaired chemotactic responsiveness of rabbit neutrophils after casein treat­ ment. Cells were treated with various concentrations of casein at 37°C for 3'A h, washed 3 times, suspended in fresh medium at 2°C for 12 h, then resuspended in fresh medium and tested in a chemotaxis experiment. Casein was used as the attractant.

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should be elucidated carefully whether de­ creased chemotaxis, induced by overoptimal stimuli, is characteristic for the recognition mechanism studied or whether instead it is of a rather artificial nature. In the case of leukocyte chemotaxis assayed with the Boyden chamber, the following factors could account for a decreased number of cells per field when overoptimal concentrations of an attractant are used. Artifacts: (a) cytotoxicity; (b) viscosity changes, and (c) cell detachment from the filter after migration. Factor-specific effects leading to de­ creased migration: (a) impaired recognition of the diffusion gradient; (b) impaired rec­ ognition of the substance itself, and (c) in­ duction of migration inhibition, be it due to metabolic changes or to alteration of surface adhesiveness. The data presented indicate that migra­ tion inhibition can be induced by chemotactic factors in nontoxic overoptimal concen­ trations and they further suggest that the same recognition mechanism, as for induc­ tion of directional migration, is involved. Evidence for this view was obtained by par­ allel testing of cytotaxins in overoptimal concentrations for chemotactic attraction in the Boyden chamber assay and for migra­ tion inhibition in the capillary tube assay. A dose-dependent correlation of decreased chemotaxis by overoptimal cytotaxin con­ centration and migration inhibition was ob­ served when using casein as attractant and rabbit neutrophils and macrophages as test cells. A similar behavior of rabbit neutro­ phils was observed when acid-denatured anaphylatoxin was tested. However, the latter agent acted neither as an attractant for rab­ bit macrophages nor did it influence capil­ lary tube migration at any concentration.

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Thus, cell specificity of this agent was maintained when migration inhibition in­ stead of directional migration was mea­ sured. There is good evidence that in this case, no artifact led to parallel decrease of chemotactic migration and migration inhibi­ tion. For both factors, cytotoxicity was ruled out by trypan blue exclusion tests, and the data in table I suggest that leukocytes are functionally intact if incubated in casein of inhibitory concentration for a prolonged period of time. Not even irreversible chemo­ tactic deactivation, as described by Ward and Becker [23] for other systems, was ob­ served. Other artifacts could be ruled out accordingly. However, the restriction has to be made that with both assays a possible change in cell surface leading to an altered adherence could not be excluded. Such a change could alter cell adhesiveness either to each other or to the plastic or filter material of the test chambers respectively. Since meas­ urements of surface adherence of leukocytes incubated in various cytotaxin concentrations were not performed it is not possible to de­ cide whether the observed migration inhibi­ tion in either assay represents alterations in cell surface characteristics or metabolic changes in the cells or both. Migration inhibition is a widely studied phenomenon in delayed hypersensitivity re­ actions [5, 16]. A factor (or a number of factors) derived from lymphocytes has been described which inhibits macrophage migra­ tion in vitro. Macrophage migration inhibi­ tion factor (MIF) is thought to be responsi­ ble for monocyte accumulation at sites of delayed hypersensitivity reactions [4, 5, 8, 16]. Recent data suggest that the same sub­ stance also activates macrophages [17]. Che­ motactic activity for monocytes and neutro­ phils is also present in lymphocytes superna­

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Leukocyte Chemotaxis and Migration Inhibition

oped for demonstrating migration inhibition [3, 7, 12]. Since in this report parallel re­ sults were obtained with the capillary tube technique and the Boyden chamber assay when inhibitory concentrations of chemoat­ tractants were applied, the Boyden chamber technique also offers an assay for measuring migration inhibition. Indeed, Wilkinson [25] has already measured MIF activity using a modified Boyden chamber assay.

Acknowledgement This work was supported by Swiss National Science Foundation, Grant. Nr. 3.8750.72 and by Hoffmann-La Roche AG, Basel. I thank Dr. J. H. Wissler for discussions and suggestions concerning this work, Prof. E. Sorkin and Prof. S. Normann for critical reading of the manuscript and for val­ uable discussions. The skillful technical assistance of Miss Jytte Kerschbattmer-Becli for the chemo­ taxis assay and of Miss Anna Arcon for migration inhibition assay is gratefully acknowledged. I am also grateful to Miss Helene Kreuzer for typing of the manuscript.

References 1 Adler, J.: Chemoreceptors in bacteria. Science, N.Y. 166: 1588-1597 (1969). 2 Adler, J.: A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis by Escherichia coli. J. gen Microbiol. 74: 77-91 (1973). 3 Astor, S. H.; Spitler, L. E.; Frick, O. L., and Fudenberg, H.: Human leukocyte migration in­ hibition in agarose using four antigens: corre­ lation with skin reactivity. J. Immun. 110: 1174-1179 (1973). 4 Bloom, B. R. and Bennet, B.: Mechanism of a reaction in vitro associated with delayed-type hypersensitivity. Science, N.Y. 153: 80-82 (1966). 5 Bloom, B. R. and Glade, P. R.: In vitro meth­ ods in cell-mediated immunity (Academic Press, New York 1971).

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tants, but Ward et al. [24] were able to dis­ sociate it from MIF activity. Since it could be demonstrated here that overoptimal doses of chemotactic factors of non-lymphocyte origin can display migration inhibition, it seems reasonable to assume that leukocyte accumulation in vivo could in part be regu­ lated by chemotactic factors themselves. Moreover, chemotaxis and/or migration in­ hibitors present in normal serum and plas­ ma may influence leukocyte migration as well [10,15, 22, Jungi, unpublished]. The statement that an overoptimal dose of chemotactic factor leads to migration in­ hibition is still limited to the two factors tested in this report. Not enough data are available for justifying the conclusion that decrease of chemotaxis is occurring general­ ly when a chemotactic factor is present in an overoptimal dose. It may be noteworthy that in this study, decreased chemotaxis with higher casein concentrations was not ob­ served with guinea pig neutrophils in con­ trast to the results with rabbit cells. Whether casein could induce migration inhibition in guinea pig neutrophils if applied in still higher concentrations could not be determin­ ed because of the limited solubility of casein. Further studies with a number of fairly pure and readily soluble chemotactic factors will have to show whether (a) an overoptimal dose of a chemotactic factor is generally cor­ related with a decrease of chemotactic re­ sponse, and (b) whether such a decrease is generally due to true migration inhibition. Although still widely used for measuring migration inhibition [5, 8, 9, 19, 20], the capillary tube test first introduced by George and Vaughan [11] has several dis­ advantages which make interpretation of the results difficult. In recent years, new meth­ ods such as agarose tests have been devel­

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6 Boyden, S. V.: The chemotactic effect of mix­ tures of antibody and antigen on polymor­ phonuclear leucocytes. J. exp. Med. 115: 453-466 (1962). 7 Clausen, J. E.: Migration inhibitory effect of cell-free supernatants from tuberculin-sti­ mulated cultures of human mononuclear leu­ kocytes demonstrated by two step MIF agarose assay. J. Immun. 110: 546-551 (1973). 8 David, J. R.: Delayed hypersensitivity in vitro: its mediation by a cell-free substance formed by lymphoid cell-antigen interaction. Proc. natn. Acad. Sci. USA 56: 72-77 (1966). 9 Dumonde, D. C.: ‘Lymphokines’: molecular me­ diators of cellular immune responses in ani­ mals and man. Proc. R. Soc. Med. 63: 899-902 (1970). 10 Gallin, J. I. and Kaplan, A. P.: Mononuclear cell chemotactic activity of kallikrein and plas­ minogen activator and its inhibition by C l in­ hibitor and a 2-macroglobulin. J. Immun. 113: 1928-1934 (1974). 11 George, M. and Vaughan, J. H.: In vitro cell migration as a model for delayed hypersensi­ tivity. Proc. Soc. exp. Biol. Med. I l l : 514-521 (1962). 12 Houck, J. C. and Chang, C. M.: A new sensi­ tive assay for macrophage inhibitor factor. Proc. Soc. exp. Biol. Med. 142: 800-803 (1973). 13 Jungi, T. W.: Assay of chemotaxis by a revers­ ible Boyden chamber eliminating cell detach­ ment. Int. Archs Allergy appl. Immun. 48: 341-352 (1975). 14 Jungi, T. W.: Lack of correlation between structural features and function of synthetic agents tested for leukocyte chemotaxis. Experientia (in press; 1977). 15 Keller, H. U.; Hess, M. W„ and Cottier, J.: The in vitro assessment of leucocyte chemotax­ is. Nouv. Revue, fr. Hemat. 13: 885-886 (1973). 16 Lawrence, H. S. and Landy, M.: Mediators of cellular immunity (Academic Press, New York 1969). 17 Nathan, C. F.; Remold, H. G., and David, J. R.: Characterization of a lymphocyte factor which alters macrophage functions. J. exp. Med. 137: 275-290 (1973). 18 Pick, E. and Turk, J. L.: The biological activi­

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ties of soluble lymphocyte products. Clin. exp. Immunol. 10: 1-23 (1972). 19 S0borg, M.: In vitro demonstration of microbi­ al cellular hypersensitivity in man. Proc. R. Soc. Med. 63: 903-905 (1970). 20 Sorg, C. and Bloom, B. R.: Products of acti­ vated lymphocytes. I. The use of radiolabeling techniques in the characterization and partial purification of the migration inhibitory factor of the guinea pig. J. exp. Med. 137: 148-170 (1973). 21 Stecher, V. J. and Sorkin, E.: The chemotactic activity of leucocytes related to blood coagula­ tion and fibrinolysis; in Sorkin Chemotaxis: its biology and biochemistry, pp. 362-368 (Karger, Basel 1974). 22 Ward, P. A.: The regulation of leukotactic me­ diators; in Sorkin Chemotaxis: its biology and biochemistry, pp. 333-337 (Karger, Basel 1974). 23 Ward, P. A. and Becker, E. L.: The deactiva­ tion of rabbit neutrophils by chemotactic fac­ tor and the nature of the activatable esterase. J. exp. Med. 127: 693-709 (1968). 24 Ward, P. A.; Remold, H. G., and David, J. R.: The production by antigen-stimulated lympho­ cytes of a leucotactic factor distinct from mi­ gration inhibitory factor. Cell. Immunol. 1: 162-174 (1970). 25 Wilkinson, P. C.: Chemotaxis and inflamma­ tion (Churchill-Livingstone, Edinburgh 1974). 26 Wissler, J. H.: Chemistry and biology of the anaphylatoxin related serum peptide system. I. Purification, crystallization and properties of classical anaphylatoxin from rat serum. Eur. J. Immunol. 2: 73-83 (1972). 27 Wissler, J. H.; Stecher, V. J., and Sorkin, E.: Chemistry and biology of the anaphylatoxin related serum peptide system. III. Evaluation of leucotactic activity as a property of a new peptide system with classical anaphylatoxin and cocytotaxin as components. Eur. J. Immu­ nol. 2: 90-96 (1972). 28 Wissler, J. H.; Stecher, V. J., and Sorkin, E.: Secondary structural properties of anaphyla­ toxin preparations and chemotactic activity for neutrophils. J. Immun. I l l : 314 (abstract; 1973). Correspondence to: Dr. T. W. Jungi, James A. Baker Institute for Animal Health, Cornell Uni­ versity, Ithaca, NY 14853 (USA)

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Different concentrations of chemotactic factors can produce attraction or migration inhibition of leukocytes.

Int. Archs Allergy appl. Immun. 53 : 29-36 (1977) Different Concentrations of Chemotactic Factors Can Produce Attraction or Migration Inhibition of L...
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