Chemotaxis of macrophage in inflammation

Comp. lmmun. Microbiol. inject. Dis. Vol. 8, No. 2, pp. 73-87, 1985 Printed in Great Britain

CHEMOTAXIS

OF MACROPHAGE

0147-9571/85 $3.00+0.00 Pergamon Press Ltd

IN INFLAMMATION

HIDEO HAYASHI, TEIZO YOSHIMURA a n d CHEN JINYAN* Department of Pathology, Kumamoto University Medical School, Kumamoto 860, Japan Abstract--Our particular attention in this article was given to natural mediators for macrophages isolated from the sites of tissue injury. A number of chemotactic factors, which may satisfy many criteria making them acceptable as inflammatory leucocyte chemotactic factors, has been separated. Among them, our laboratory has isolated three macrophage (monocyte) chemotactic factors (MCF-a, -b and -c). Their purification, characterization and functional specificity are discussed. Key words: chemotaxis, macrophage, monocyte, MCF-a, -b, -c, inflammation, leucocytes

CHIMIOTACTISME DES MACROPHAGES DANS LES INFLAMMATIONS R6sum~-Dans cet article, l'auteur s'attache/t l'&ude des m6diateurs naturels du chimiotactisme des macrophages isol6s de 16sions tissulaires. De nombreux facteurs chimotactiques, ob6issant aux crit6res qui permettent de les classer parmi les facteurs chimiotactiques des leucocytes inflammatoires, ont 6t6 s6par6s. Parmi eux, trois agissent sur le macrophage (monocytes): MCF-a, - b e t -c. Leurs purification, caract6risation et sp6cificit6 fonctionnelle sont discut6es. Mots-clefs: chimiotactisme, macrophage, monocytes, MCF-a, -b, -c, inflammation, leucocytes

INTRODUCTION

As is well known, in many types of non-immunologically and immunologically mediated inflammations, a number of macrophages are found at extravascular locations of the lesions with other types of leucocytes; and it has been shown that monocytes in the circulation are precursors of the effector macrophages, and emigrate into the lesions by the effect of chemotactic factors which are locally produced. Macrophage chemotactic factors produced in vitro, including lymphokines [1-4] and complement products [5], have been described. However, chemical factors capable of inducing their selective emigration to inflammatory sites have not been satisfactorily elucidated. Accordingly, separation and characterization of macrophage chemotactic factors present in inflammatory tissues or exudates are strongly required for essential understanding of the mediation of macrophage response. Cohen et al. [6] have found two macrophage chemotactic factors in skin extract from guinea-pigs undergoing hypersensitivity reactions to bovine ~-globulin or egg albumin, or contact hypersensitivities to O-chlorobenzoyl chloride; one is sedimented in the region of an IgG marker, and the other sedimented near an albumin marker. The former resembles chemotactic lymphokine found in supernatant of O-chlorobenzoyl chloride-sensitized lymphoeytes cultured with specific antigen. Postlethwaite and Snyderman [7] have detected macrophage chemotactic lymphokine with a mol. wt of about 12,500 in guinea-pig *Present address: Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China. 73

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peritoneal exudate induced by challenge with horseradish peroxidase. Kambara et al. [8] and Ueda et al. [9] have found three macrophage chemotactic factors in bovine 7-globulin-induced hypersensitivity skin lesions in guinea-pigs; the major chemotactic factor seems to resemble a macrophage chemotactic factor with a mol. wt of about 200,000 detected in vitro in guinea-pig serum [10] because their chemotactic factor is derived from serum protein (possibly from fl-globulin) but not from lymphocytes. I. NATURAL MEDIATORS FOR MACROPHAGE (MONOCYTE) EMIGRATION A. IgG-derived macrophage chemotactic factor (MCF-a, monoegresin)

Macrophages accumulated in the skin lesions are characterized by many lysosomal granules which stained for acid phosphatase [11, 12] and counted quantitatively [13]. Macrophage reaction reaches its peak in about 24h in either DNP-ascaris extract (DNP-As)- or PPD-induced skin lesions in the guinea-pigs sensitized with DNP-As or BCG, respectively (Fig. la, b); and chemotactic activity of these skin extracts for guinea-pig peritoneal exudate macrophages and blood monocytes clearly parallels the time course of macrophage response [12, 14]. Three different macrophage chemotactic factors a, b, and c (MCF-a, -b, and -c) can be respectively separated from the skin extracts by gel filtration followed by chromatography using DEAE-Sephadex. MCF-a (monoegresin) is further purified by chromatography with CM-Sephadex followed by immunoadsorbent chromatography with anti-IgG antibody [14, 15] (Table 1). This chemotactic agent, recovered from the immunoadsorbent columns in an acid condition, is a heat-labile (56°C for 30 min) protein, and has an approximate mol. wt of 150,000 when measured by gel filtration. Intradermal injection of MCF-a at a reasonable amount into guinea-pigs provokes marked macrophage accumulation into the treated sites; it causes no vascular permeability change and hemorrhagic change. (a)

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Fig. l. Time-course of leucocyte emigration in cutaneous allergic inflammation in guinea pigs. (a), Inflammation was induced by intradermal injection of 250/~g DNP-As; (b), it was induced by intradermal 10~g PPD. Macrophages were differentiated from lymphocytes histologically and histochemically; the ceils were characterized by many cytoplasmic granules stained for acid phosphatase. • • , Neutrophil reaction; n - - - u , macrophage reaction; • . . . . O, lymphocyte reaction. Emigrated cells were counted according to Kay [13].

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Table 1. Procedures of isolation and purification of macrophage chemotactic factors (MCFs)* DNP-As-induced or PPD-induced skin lesions 50-,um slices powdered with cold acetone, extracted with 0.067 M phosphate buffer (pH 7.4) at 4:C for 4 h

Extract (SPA = 101) Eluted in 0.067 M phosphate buffer (pH 7.4) on Sephadex G-100

I First peak (SPA - 189) Eluted in phosphate buffer

Second peak (SPA = 99) M C F - b (mol. wt = 14,000)

(pH 7.4) on DEAE-Sephadex A-50

r Eluted in 0.02 M

Eluted in 0.3 M

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(SPA = 540)

Eluted in the first peak of linear gradient elution with NaC1 on CM-

Eluted in the second peak of linear gradient elution with NaCI on CM-Sephadex

Sephadex C-50 (pH 6.8) (SPA = 1120)t

C-50 (pH 5.5) (SPA = 2645)

Eluted on anti-IgG affinity column followed by elution with 1.0 M acetic acid (pH 2.4) (SPA - 2700)

Fractionated with 0.025 M aminocaproic acid Tris buffer (pH 8.6) by isotachophoresis (SPA = 6250)~

M C F - a (mol. wt = 150,000)

MCF-c (mol. wt = 110,000)

*SPA, specific activity, numbers of macrophages migrated/E280 nm. Molecular weight estimated by gel filtration.

t l f necessary, this step was repeated until the elution profile became homogeneous. ~Second fraction (MCF-c) specific for macrophage chemotaxis; fourth fraction (LCF-d) for lymphocyte chemotaxis.

MCF-a shares common antigenicity with guinea-pig IgG; its chemotactic activity is completely absorbed by anti-IgG and anti-light chain antibodies, but not by anti-C5 and anti-MCF-c antibodies. It is of particular interest to note that MCF-a is active for macrophages but not for neutrophils in vitro and in vivo, while neutrophil chemotactic factor (leucoegresin) (reviewed by Hayashi et al. [16] and Hayashi [17]), which was generated from IgG molecule by mild digestion with neutral thiol protease released by antigen-stimulated resident macrophages [18] or by immune complex-stimulated neutrophils [19], and separated from inflamed skin is active for neutrophils but not for macrophages in vitro and in vivo. Accordingly, it seems reasonable that the thiol protease fails to generate in vitro macrophage chemotactic activity from IgG molecule. Based on the observations that MCF-a shares a common antigenicity with IgG, and is active for macrophages but not for neutrophils and that macrophage response in the skin lesions (Fig. 1) occurs after neutrophil emigration, it has been demonstrated that this type of macrophage chemotactic agent is generated from IgG molecule by a certain neutral protease from neutrophils, which is different from the above thiol protease. The enzyme is separated at acid pH from rabbit peritoneal exudate neutrophil granules, and purified by elution on DEAE-Sephadex, CM-Sephadex, and Sephadex G-75 in that order; it has a mol. wt of about 27,000 estimated by SDS-PAGE, and is found to be high cationic charged isomers (pI 10.1 and pI 10.9) by isoelectric focusing technique, and optimally active at pH 7.0 when tested against hemoglobin (3HHb) [20,21]. This enzyme is

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HIDEO HAYASHI et al.

considered to be a serine protease resembling neutrophil elastase, especially human neutrophil elastase, because of strong inactivation by diisopropyl fluorophosphate, phenylmethyl sulfonylfluoride, soybean trypsin inhibitor or elastatinai and of similar kinetics in synthetic substrate digestion [21]. B. C5-Derived macrophage chemotactic factor (MCF-b ) As described above, MCF-b is extracted from 24-h DNP-As-induced skin lesions in guinea-pigs (Fig. la), and partially purified by gel filtration; it is heat-stable and has an approximate mol. wt of 14,000. Chemotactic activity of MCF-b is completely absorbed by anti-C5 antibody, but not by anti-C3, anti-IgG, or anti-MCF-c antibody [14, 22]; and immunoadsorption with anti-C5 antibody induces about 30~o decrease in the macrophage chemotactic activity of the skin extract. Further study demonstrates that complement depletion induced by cobra venom factor eliminates about 30~o of macrophage chemotactic activity of skin extract, and decreases considerably the intensity of macrophage reaction [22]; and no decrease in the activity of the skin extract is detected after immunoadsorption with anti-C5 antibody. The observations strongly suggest involvement of complement system such as C5 in the mediation of macrophage reaction in the model inflammation (Fig. la). A heat-stable macrophage chemotactic factor with a mol. wt between 13,000 and 15,000 has been derived in vitro from C5 when guinea pig serum was treated with immune complexes, endotoxin, or cobra venom factor [3, 23]. Another heat-stable macrophage chemotactic factor with similar molecular weight has been generated in vitro from C5 when digested with neutral chymotrypsin-like enzyme with a mol. wt of about 35,000 from neutrophil granules [24]. In view of observations that MCF-b closely resembles the proteolytic product of C5 and that macrophage accumulation occurs after neutrophils in the skin lesions, it seems reasonable that MCF-b may be produced in vivo by the neurophil granular protease from the C5 molecule. As discussed below, macrophage reaction in DNP-As-induced skin lesions may mostly be associated with the combined action of MCF-a and -b; MCF-a is more important than MCF-b, and MCF-c is apparently less important, while that in PPD-induced skin lesions mostly associated with MCF-a and -c; MCF-c is more significant thant MCF-a, and MCF-b is apparently less significant. C. Lymphocyte-derived macrophage chemotactic factor ( M CF-c ) MCF-c is extracted from skin lesions induced by DNP-As and PPD in guinea-pigs (Fig. 1). By gel filtration followed by chromatography with DEAE-Sephadex, this chemotactic agent can be separated from MCF-a and -b. MCF-c is highly purified by chromatography with CM-Sephadex followed by preparative isotachophoresis, as confirmed by capillary isotachophoresis technique [15] (Table 1); it is heat-stable and has an approximate mol. wt of 110,000 and isoelectric point of 5.2. This agent exhibits marked macrophage chemotactic activity but no neutrophil chemotactic, lymphocyte chemotactic, and MIF activity in excess of that in vitro. It is of importance to note that chemotactic activity of MCF-c is completely absorbed by anti-MCF-c antibody but not by anti-IgG, anti-C5 or anti-C3 antibody, indicating that MCF-a, -b, and -c are different from each other in the antigenicity. Its chemotactic activity absorbed on the immunoadsorbent column is eluted in acid without loss of activity. The

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anti-MCF-c antibody is further demonstrated & vitro to have no anti-MIF, anti-neutrophil chemotactic, or anti-lymphocyte chemotactic activity. It is of further interest that approximately 70~ of macrophage chemotactic activity, released in vitro from sensitized guinea-pig lymphocytes stimulated by PPD, can be absorbed by anti-MCF-c antibody but not by anti-IgG or anti-C5 antibody, indicating that MCF-c shares common antigenicity with the major macrophage chemotactic lymphokine [15]. This lymphokine absorbed on the immunoadsorbent column is eluted in acid without loss of activity, and successfully detected as a single band on polyacrylamide gel electrophoresis; it is heat-stable and has a mol. wt of approximately 12,500, identical with that of macrophage chemotactic lymphokine previously described. The minor macrophage chemotactic lymphokine, which has a mol. wt similar to that of bovine serum albumin and associates with the remaining 30~o of the chemotactic activity released by PPD-stimulation, is unable to detect in the skin lesions mentioned above (Fig. 1). Subsequent study has shown that anti-MCF-c antibody can absorb almost completely macrophage chemotactic activity released by sensitized guinea pig lymphocytes stimulated by bovine-~-globulin and horseradish peroxidase, or by nonsensitized lymphocytes stimulated by PHA; these lymphokines, eluted from the immunoadsorbent columns in acid, yield similarly a single band on polyacrylamide gel electrophoresis, and are indistinguishable from PPD-lymphokine in respect of chemotactic activity, physicochemical property, and antigenicity [17, 25]. It has been demonstrated the MCF-c exists in the form of complexes with some serum protein (possibly c~-globulin) exuded by the mechanism of increased vascular permeability at the skin site. The evidence for this suggestion is that MCF-c shares common antigenicity also with guinea-pig serum, and that MCF-c can be dissociated from its complex with serum protein without loss of activity under acid condition; the dissociated active agent closely resembles macrophage chemotactic lymphokine. Further observations have demonstrated that the chemotactic lymphokine eluted behaves as a higher molecular substance in the presence of guinea-pig serum, suggesting that the lymphokine may form a complex with serum protein at neutral pH; and the complex of that lymphokine and serum protein is dissociated by acid condition without loss of its activity [25]. Intradermal injection of MCF-c into normal guinea-pigs induces marked mononuclear cell reaction; the cells are distributed perivascularly (it causes no vascular permeability change and hemorrhagic change). No demonstrable change of the vessel walls is observed. Little emigration of cells other than mononuclear cells is revealed, suggesting the functional specificity of MCF-c for macrophages; most of the cells emigrated are characterized by many cytoplasmic granules, which stained for acid phosphatase. It is of importance that intradermal injection of the in vitro complex induces mononuclear cell reaction in a similar pattern, while that of the lymphokine in the free form provokes apparently less marked mononuclear cell reaction, suggesting the biological role of the serum protein as a carrier protein. Further study has shown that the above lymphokines, produced by stimulation with bovine 7-globulin, horseradish peroxidase or PHA, also bind in vitro with some serum protein(s) under neutral condition, but the complexes formed are dissociated under acid condition. It may be apparent that macrophage chemotactic lymphokine bound to carrier molecule(s) is responsible for the biological macrophage chemotactic activity in delayed hypersensitivity reaction site. The determination of serum protein(s) to bind with the lymphokines is strongly required. Regarding to the T cell subset producing macrophage chemotactic lymphokine, Miura

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et al. [26] have shown that mouse macrophage chemotactic lymphokine can be primarily

produced by mouse Lyt 1+ 2- T cell subset when stimulated by dinitrophenyl ovalbumin or Con A at low concentrations, utilizing monoclonal anti-Thy 1.2, anti-Lyt 1.1, or anti-Lyt 2.1 antibody. The Lyt 1+ 2- T cell population has been reported to be primarilly responsible for MIF production [27, 28] and lymphotoxin secretion [29]. Roehm et al. [30] have demonstrated that the T cell population provokes a significant lymphocyte-induced angiogenesis, which may be attributed to an effect mediated by lymphokine. Furthermore, production of macrophage chemotactic lymphokine by animal lectin of rat has been described. This animal lectin with a mol. wt of 70,000 has been separated from differentiated rat ascites hepatoma cell surface, and highly purified by chromatography (reviewed by Hayashi and Ishimaru [31]. The glycoprotein induces not only aggregation of the cells but also adhesiveness characterized by development of junctional complexes; it is synthesized by the cells but not by undifferentiated rat ascites hepatoma cells. The adhesive lectin possesses a mitogenic activity on rat T lymphocytes [32]; and T lymphocytes activated by the lectin produce macrophage chemotactic lymphokine and release extracellularly [33]; the lymphokine is heat-stable and has an approximate mol. wt of 12,500, closely resembling macrophage chemotactic lymphokines described above. The kinetics of its production are clearly comparable with those of production of human lymphotoxin generated by mixed lymphocyte culture [34] and human monocyte chemotactic lymphokine produced by Con A-stimulation [35]. It has been suggested that the lymphokine produced by the adhesive lectin may play a role in the mediation of intense mononuclear cell reaction in the site of differentiated hepatoma cell proliferation [31, 33]. In contrast, mononuclear cell reaction in the site of undifferentiated hepatoma cell proliferation is apparently less marked, indicating no involvement of the lymphokine. The differentiated hepatoma cells slowly proliferate in the site, while the undifferentiated hepatoma cells rapidly proliferate in the site, suggesting the biological role of macrophages accumulated. II. ACTIVITY DYNAMICS OF MACROPHAGE CHEMOTACTIC FACTORS I N VIVO It has been demonstrated that macrophage chemotactic activity in extract from 24-h DNP-As-induced skin lesions (Fig. 1a) is primarily associated with MCF-a and -b, because approximately 90~o of the chemotactic activity can be absorbed by the combined use of these antibodies; the approximate activity ratio of MCF-a and -b in the extract is 60:30, chemotactic activity of MCF-c is apparently weak [14]. On the other hand, the same immunoadsorbent chromatography using the antibodies has revealed that chemotactic activty of MCF-a, -b and -c in extract from 24-h PPD-induced skin lesions (Fig. lb) manifests an approximate ratio of 35:5:60, indicating that most of macrophage chemotactic activity are associated with MCF-a and -c and that MCF-c may play a greater role than MCF-a in the mediation of the macrophage reaction; MCF-b activity is almost negligible [15]. Subsequent experiments have shown that chemotactic activity of MCF-a and -c in early extract from 6-h PPD-induced skin lesions (Fig. lb) represents an approximate ratio of 70:20, suggesting that MCF-a may be more significant than MCF-c in the mediation of early macrophage reaction [17, 36]. The observations have strongly suggested that the increase or decrease in the chemotactic activity of each MCF is parallelly associated with the intensity in the accumulation of neutrophils and lymphocytes. As mentioned above,

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since production of MCF-a and -b is associated with respective proteases from neutrophils emigrated, these agents may be called "neutrophil-associated macrophage chemotactic factors", while since MCF-c is produced by sensitized lymphocytes emigrated, it may be called "lymphocyte-associated macrophage chemotactic factor". The heterogeneity of each MCF and the dynamic change in their activities, which are associated with the process of macrophage accumulation in the skin lesions, strongly suggest the functional specificity of each MCF for macrophages. This is obviously a matter of importance for future investigation. III. FUNCTIONAL SPECIFICITY OF MACROPHAGE CHEMOTACTIC FACTORS Mouse macrophages appear to be most favorable for determining the functional specificity of each MCF because of their better establishment on the functions and cell surface markers. Fortunatley, there exists no restriction between the MCFs and macrophages (monocytes) from guinea-pigs and other species [37, 38]. All of the cells tested respond to three kinds of MCFs; human blood monocytes react with the MCFs more strongly than guinea pig peritoneal exudate macrophages and blood monocytes, while the responsiveness of mouse peritoneal exudate macrophages to each MCF is much lower than that of guinea-pig cells. In view of observations mentioned above, functional specificity of MCF-a, -b and -c has been first assayed using cell line cells of macrophages as follows [37, 38]. M~ cells [39], which are myeloid leukemic cell line cells established from a SL/AM mouse (H-2 q) [39], differentiate into macrophage-like cells, Mi~, when they are cultured with conditioned medium obtained from the secondary culture of murine embryonic fibroblasts in vitro [40, 41]. Mi~ cells express Fc receptor-dependent phagocytic activity, although weak, and accessory cell (A cell) function for lymphocytes in association with loss of proliferating activity [42] but with appearance of Ia antigens [41]. Two kinds of sublines are further established; Mm~ cells, a subclone of M j cells, are highly phagocytic, but lack both A cell activity and Ia antigen [42], and Mk~ cells, a subclone of Mi~ cells, are poorly phagocytic in spite of the presence of Fc receptor, but possess A cell activity and Ia antigen. Thus, these cell line cells, especially Mk~ and Mmt cells, show a conspicuous contrast in functions as macrophages [42]. Mm~ cells respond to MCF-a and -b, but do not migrate toward MCF-c (Fig. 2). Conversely, Mkl cells migrate only toward MCF-c but not toward MCF-a and -b (Fig. 2). The development of chemotactic responses to MCF-c seems to be accompanied by appearance of Ia antigen on the cells. Cytotoxic treatment of M~+ cells, cocultured with conditioned medium for 72 h, with anti-Ia antisera plus complement results in the specific decrease of responsiveness to MCF-c, while leaving those to MCF-a and -b. Furthermore, MCF-c attracts Ia positive Mi~ cells, while MCF-a and -b attract Ia negative M( cells. Thus, the results indicate the existence of two migrating subpopulations of M + cells with specificity for MCF-a and -b, or for MCF-c, respectively. Thus, it has been suggested that MCF-c attract chemotactically Ia-bearing accessory macrophages, and MCF-a and -b Ia-negative macrophages in the hypersensitivity skin lesions induced by PPD. It has been further demonstrated that macrophage chemotactic lymphokines from Con A-stimulated lymphocytes of mouse and guinea-pig similarly attract Mk~ cells but not Mm~ cells [43]. Belier and Ho [44] have shown that most of elicited and resident peritoneal macrophages can be converted to Ia-positive in vitro in response to a lymphokine produced by activated

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T cells; the latent period before actual Ia expression varies from 3 to 7 days, depending on the target population. Since transition from Ia-negative M + cells to Ia-positive cells is not induced during 8-day-culture in the presence or absence of MCF-c, the Ia-negative cells are not converted to Ia-positive cells in the time of chemotaxis by MCF-c, suggesting that MCF-c may be different from the activating agents which induce the conversion of macrophages form Ia-negative to Ia-positive. It has further been demonstrated that chemotactic response of the Ia-positive cells by MCF-c is not influenced by previous treatment of the cells with monoclonal anti-Ia antibody, suggesting that the receptor site for M C F - c on the cell surface may be different from the Ia antigen site. It has been reported that a human monocyte-like cell line U937 develops the ability to respond chemotactically t o / n vitro macrophage chemotactic factors when cultured in the presence of lymphokines [45, 46]. The chemotactic response to zymosan-activated human serum and macrophage chemotactic lymphokine develops earlier than does the response to fMet-Leu-Phe [46], although the difference in the chemotactic properties of these factors is remained to be ascertained. In additon, the development of chemotaxis to fMet-Leu-Phe during differentiation of U937 cells has been described to be associated with simultaneous appearance on the cells of specific surface receptors for the chemoattractant [45], suggesting that the induction of chemotaxis to both M C F - a and -b, and M C F - c presumably require certain membrane changes of the M { cells during differentiation. It has been further suggested that MCF-a, -b and -c may be recognized by different receptors and that most of responding cells do not have all three receptors (Honda et al., unpublished). The results appear to contrast with studies showing that the great majority of responding human monocytes have all three receptors to the following in vitro macrophage chemotactic factors such as C5a, macrophage chemotactic lymphokine, and fMet-Leu-Phe [47]. Major reasons which account for the difference between these studies may result from that of in vitro assay system, such as responding cells or macrophage

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chemotactic factors. Further experiments are needed to investigate the different receptor specificities on the M( cells or monocytes such as the binding experiment of the radiolabeled macrophage chemotactic factors [45, 46]. Thus, although MCF-c respond to specific chemotactic receptor on the Ia-positive M + cells, no correlation between blocking studies of Ia antigen and chemotactic activity indicates that there is no interaction between MCF-c and Ia antigen on the surface of M[ cells during the time of chemotaxis. Chemotactic response to various macrophage chemotactic factors of other macrophage cell lines has been reported. J774.1 exhibits good chemotaxis to endotoxin-activated mouse serum, and very poor chemotaxis to macrophage chemotactic lymphokine [35]. Another four of the cell lines (RAW 246, RAW 309CR, PU5-IR WR 19M.I) exhibit similar chemotactic response [48]. In this respect, Mmj cells resemble the 5 cell lines. On the other hand, P388D1 cells exhibit no chemotaxis to C5a but respond to endotoxin-activated serum or macrophage chemotactic lymphokine [35]. WEHI-3 exhibits no chemotaxis to C5a but respond to endotoxin-activated serum [48]. In view of observations that Mk~ cell is a new cloned macrophage-like cell line, posessing Ia antigen and A-cell activity [42], it is of special interest that the Mkt cells are capable of a chemotactic response to MCF-c (complex of macrophage chemotactic lymphokine and carrier protein), whereas very poorly respond to take off MCF-a and -b. In subsequent experiments, peritoneal exudate macrophages, collected from Listeria monoeytogenes-infected mice, are separated into Ia-positive and -negative macrophages by fluorescence-activated cell sorter (FACS) analysis using anti-mouse Ia monoclonal antibody [49]; this infection preferentially increases the Ia-positive macrophages [50]. Interestingly, striking differences in responsiveness to MCFs are also confirmed on the macrophage populations (Table 2); Ia-negative populations respond to MCF-a and -b, and are virtually unresponsive to MCF-c, while Ia-positive populations respond to MCF-c, and are devoid of activity of MCF-a and -b [51]. A comparison of the responsiveness of cells previously treated with anti-Ia antibody and non-treated cells shows that prior incubation of the cells with the antibody has little effect on the chemotactic activity of the cells to MCFs. It has been demonstrated that MCF-c, when given intraperitoneally, causes selective accumulation of Ia-positive macrophages, while MCF-a and b induces selective accumulation of Ia-negative macrophages (Fig. 3) [51]. It has been further demonstrated that transition from Ia-negative macrophages to Ia-positive macrophages is not induced in the presence or absence of MCF-c during 8 days Table 2. Chematotactic comparison of MCFs for la-positive and Ia-negative macropbages Chemotactic factors MCF-a MCF-b MCF-c MCF-LDt MCF-LD:~ Medium

Unsorted macrophages* mixture 66 75 111 42 40 5

Sorted macrophages* laIa ÷ 75 79 9 10 12 6

12 13 84 40 42 4

*Plastic dish-adherent cells from peritoneal exudates of Listeria monocytogenes-infected C3H/He mice were treated with anti-la k (2) before application to FACS, tMacrophage chemotactic lymphokine from Con A-stimulated guinea pig lymphocytes. :~Macrophage chemotactic lymphokine from Con A-stimulated mouse lymphocytes.

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Fig. 3. The kineticsof Ia-positive macrophageaccumulationin the peritoneal cavityin response to 1/~g of MCF-c injected into normal mice. Values are the mean of four experiments. of culture at 37°C, indicating that over the time period (1.5 h) of the chemotactic assay, the surface Ia antigen expressed by a given cell is a stable characteristic of that cell. On the other hand, several studies have shown that Ia antigen expression of macrophages is regulated by lymphokines. Steeg et al. [52] have reported that the macrophage Ia antigen regulatory mediator in vitro shares common antigenicity and biochemical characteristics with immune interferon (INF-7). Scher et al. [53] have described that intraperitoneal injections of culture supernatants of Listeria monocytogenes-stimulated lymphocytes produce peritoneal exudates capable of enriching Ia-positive macrophages in number. Beller and Ho [44] have shown that macrophages that were Ia-positive when separated spontaneously lose the Ia antigen in culture; however, in the presence of lymphokine most of the macrophages can be induced to express Ia antigen. These observations have suggested that there is an interconversion of Ia-positive and Ia-negative phentotypes, and the Ia-positive and Ia-negative macrophage subpopulations are not stable subsets. Experiments in progress (Honda et al., unpublished) have demonstrated that purified MCF-c is clearly distinct from IL-2 and IFN-~. As mentioned above, MCF-c differs from lymphocyte chemotactic lymphokines, neutrophil chemotactic lymphokines, MIF, macrophage activating factor, leucocyte inhibitory factor, or lymphotoxin [25]. However, the observations do not preclude the possibility that MCF-c acts synergistically with the IFN or other Ia antigen-expressing agent. It has also been demonstrated that the macrophage populations attracted by MCF-c assist active antigen-presenting function, while those attracted by MCF-a and -b do not. Further interest is in the chemotactic responsiveness of dendritic cells characterized by Steinman and Cohn [54]. However, the cells with characteristics of dendritic cells are only very few among the non-phagocytic Ia-positive adherent cells in peritoneal exudates of Listeria monocytogenes-infected mice. Using mouse dendritic cell line (KS) established by Inaba et al. [55], Honda et al. (unpublished) have found that KS cells migrate towards none

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of the MCF-a, -b and -c, indicating that the Ia-positive cells attracted by MCF-c are regarded as Ia-positive macrophages but not dendritic cells. As mentioned above, since MCF-a, -b and -c were isolated from PPD-induced skin lesions in guinea-pigs, it becomes crucial to establish the nature of the functional specificity of guinea pig macrophages for the MCFs. For detecting the Ia antigen, strain 2 anti-strain 13 serum (anti-Ia 1, 3, 7) and strain 13 anti-strain 2 serum (anti-Ia 2, 4) have been used. The guinea pig Ia antigens have been reported to be expressed on subpopulations of inbred strain 13 (GPLA Ia 1, 3, 5, 6, 7) and strain 2 (GPLA Ia 2, 4, 5, 6) macrophages and that such subpopulations may play a critical role in the activation of T cell proliferation to soluble protein antigens and to ailoantigens [56]. Using Ia-positive and negative macrophages from peritoneal exudates of Listeria monocytogenes-infected guinea-pigs, Honda et al. [38] have shown that positive immunofluorescence staining with the antibodies is detected for almost all of the macrophages migrated towards MCF-c, whereas for less than 25~o of the cells migrated towards MCF-b, and less than 28~o towards MCF-b, indicating that guinea-pig peritoneal macrophages responding to MCFs consist of two distinct subpopulations, i.e. Ia-positive and Ia-negative macrophages. Based on the observations described above, the kinetics of macrophage accumulation in relation to the chemotactic activity for the cells have been analysed. Time course of macrophage and lymphocyte reactions in the peritoneal cavity of strain 13 guinea pigs challenged intraperitoneally with PPD is essentially similar to that of the cell reactions in PPD-induced skin lesions [15]; most (80-90~) of macrophages accumulated at early stage (6 h) of inflammation are Ia-negative cells, and Ia-positive macrophages markedly increase in number at later stage (12-24 h) of inflammation; most (95~o) of macrophages accumulated being Ia-positive cells (Fig. 4) [57]. Thus, it has been assumed that macrophage response in delayed-type hypersensitivity reaction in guinea-pigs appears biphasic in the chemotactic mediation, the early being associated with Ia-negative macrophages attracted by MCF-a and -b, and the latter with Ia-positive macrophages attracted by MCF-c. In subsequent study [57], it has been observed that Ia-positive monocytes are increased in number in peripheral blood of guinea-pigs immunized with BCG or Listeria monocytogenes; Ia-positive monocytes migrate towards MCF-c, and Ia-negative monocytes towards MCF-a and -b (Table 3), suggesting that a considerable number of

Time 01¢) after chsien~l

Fig. 4. Kineticsof Ia-positive macrophagesdetected after intraperitonealinjection of 10#g PPD into immunized guinea-pigs. Data represent the mean number of exudate macrophages and of Ia-positive macrophages at different intervals after injection into six animals. The number of Ia-positive macrophageswas calculatedfrom their percentagefor exudate macrophages.(O), total macrophages; (©) Ia-positivemacrophages.

84

HIDEO HAYASHI et al. Table 3. Comparative effects of Ia-positive and la-negative blood monocytes Chemotactic factors MCF-at MCF-b++ MCF-c§ PUll PF f Buffer**

No. of monocytes migrated*

% la+-monocytes migrated*

103 __+6 97 __4 108 + 5 102 + 5 22 + 4 4+ 2

15.6 + 1.8 13.5 + 1.2 87.4 + 3.3 84.6 + 2.9 15.3 + 1.5 not tested

*Mean values of five experiments in duplicate; + SUM. 3"After rechromatograpby with CM-Sephadex C-50 [15]. :~After gel filtration on Sephadex G-100 [15]. §After preparative isotachophoresis [15]. []From 24-h peritoneal exudates of animals immunized and challenged with 10 #g PPD. ¢From 24 h peritoneal exudates of animals not immunized but injected with 10#g PPD. **0.15M PBS, pH 7.2. Table 4. Immunoadsorption of MCA in PF by anti-MCF-c antibody Samples tested PF, normal PF, non-immunized but injected (24 h)* PF, immunized but not challenged++ PF, immunized and challenged (1 h)§ PF, immunized and challenged (24 h)§ Before adsorption After adsorptionll After acid elution~ Buffer**

No. of migrated macrophages* 13 _+ 3 28 _+ 2 13 _+ 4 18 _+ 5 110 _+ 9 22 _+ 2 94 _+ 6 8+ 2

*Mean values of five experiments in duplicate; _+ SUM. ?From animals not immunized but injected with 10/~g PPD; 24 h after injection. ++From animals immunized but not challenged with PPD. §From animals immunized and challenged with 10#g PPD; 1 or 2 4 h after challenge. [IPassed through immunoadsorbent column coupled with anti-MCF-c antibody. ~Fraction absorbed on the immunoadsorbent column was eluted in 1.0 M acetic acid, pH 2.4. Chemotactic activity was assayed after dialysis against 0.t5 M PBS, pH 7.2. **0.15M PBS, pH 7.2.

Ia-positive macrophages found at the inflammatory site may be those derived from Ia-positive monocytes extravascularly attracted by MCF-c. In fact, in parallel to the increased accumulation of Ia-positive macrophages in the peritoneal exudates of guineapigs, the activity of MCF-c of the peritoneal fluids was clearly increased (Table 4) [57]. However, there remains a question of whether the mediator(s) capable of inducing the transition of Ia-negative macrophages to Ia-positive macrophages, may be present in the inflammatory site. This is obviously a matter of importance for future investigation. IV. CONCLUDING REMARKS As reviewed by Hayashi et al. [58] the earliest cellular responses to inflammatory or immunological stimuli inlcude margination of leucocytes within small blood vessels at the site of the lesions; adherence of these leucocytes to the vascular endothelium, particularly in the venules; migration of adherent leucocytes between the endothelial cells and out of

Chemotaxis of macrophage in inflammation

85

the vessels; detachment of the cells and their subsequent homing onto the site of injuries. These events require two fundamental properties of cells, adhesion and locomotion, both of which present complex problems as general phenomenon as well as in the special context of inflammation. The adhesion of leucocytes to the vascular endothelium may result from an enhanced affinity of the surface of the two cells for one another. The locomotion of leucocytes is difficult to study in vivo, but has been much studied in vitro, where it can be shown to be directional or chemotactic. The cells of interest in inflammation are neutrophils, eosinophils, macrophages (monocytes), and lymphocytes. Lymphocyte emigration has often been considered to present a special case. However, it is now considered that it bears a closer resemblance to the emigration of the other cells. Accordingly, the problem of why a single chemical substance from inflammatory tissues can induce such a morphological sequence in the respective leucocyte emigration was of particular importance to clarify. In our laboratory, a number of chemotactic factors, which may satisfy many criteria making them acceptable as inflammatory leucocyte chemotactic factors, has been separated; their purification, characterization, and functional specificity has been discussed. Most of them were separated from the sites of inflammatory lesions resulting from immune responses as experimental models. Our particular attention was paid on three neutrophil chemotactic factors, three macrophage (monocyte) chemotactic factors (MCF-a, -b and -c), four lymphocyte chemotactic factors (LCF-a, -b, -c and -d), and three eosinophil chemotactic factors. It was of special importance that highly purified chemotactic factors among them exhibited cell-type-specific functions for the associated leucocytes in vitro as well as in vivo; intradermal injection of these purified chemotactic factors clearly provoked adherence of the associated leucocytes to the vascular endothelium of the venules, followed by selective emigration of the cells into the perivascular locations. Thus, it seemed acceptable that these chemotactic factors were identifiable in inflammatory lesions, and attached the cells into them. It was also reasonable that such cellular emigration could be dissociated' from enhancement of vascular permeability, because these purified chemotactic factors exhibited no vascular permeability-increasing potency. Cinemicrophotographic and ultrastructural observations on neutrophil emigration induced by leucoegresin (reviewed by Hayashi et al. [16] and Hayashi [17] have given one direction to the analysis of morphological sequence in the emigration of other leucocytes in vivo. However, the question of whether accumulation of leucocytes into inflammatory tissues is induced only by "chemotaxis" phenomenon still remains to be clarified; there may be many reasons [59]. It was also of importance that MCF-a and -b specifically attracted Ia-negative macrophages, and MCF-c did Ia-positive macrophages; LCF-a specifically attracted B lymphocytes, and LCF-b, -c and -d did T lymphocytes, suggesting the functional specificity of these chemotactic factors and the diversity of these cells. This line of research may open a new way for essential understanding of the role of the cells accumulated into inflammatory sites; the diversity in the functions of the cells accumulated may concern the biological characteristics of inflammatory tissue injuries. It was further postulated that immunoglobulin molecules, besides their function as antibodies, may play an immunologically nonspecific part as precursors of leucocyte chemotactic factors in inflammation, suggesting the dynamic metabolism of immunoglobulins under pathological conditions [17]. They included leucoegresin for neutrophil chemotaxis, monoegresin (MCF-a) for macrophage chemotaxis, and lymphoegresin (LCF-a) for lymphocyte chemotaxis; IgG molecules, exuded by the mechanism of C.I M I.D. g'2--B

86

HIDEO HAYASHI et al.

i n c r e a s e d v a s c u l a r p e r m e a b i l i t y in i n f l a m m a t i o n , f a c t o r s specific f o r n e u t r o p h i l s , m a c r o p h a g e s , proteolytic conditions.

were locally converted to chemotactic or lymphocytes under characteristic

As described above, our particular attention in this article was paid on natural mediators f o r m a c r o p h a g e s i s o l a t e d f r o m t h e sites o f t i s s u e i n j u r y . A c c o r d i n g l y , c h e m o t a c t i c f a c t o r s f o r o t h e r t y p e s o f l e u c o c y t e s w e r e n o t d i s c u s s e d in t h i s article. Acknowledgements--iThe authors are very grateful to Drs M. Honda and K. Miura for their thankful collaboration. The investigations referred to in this article were fruitfully supported by grants from the Japanese Ministry of Education, Science ~,nd Culture, and Naito Foundation, Tokyo, and the Society for Metabolism Research, Tokyo. Our thanks are also due to Miss Y. Hoshiko for typing the manuscript.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41, 42. 43.

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