Leukocyte recruitment by monocyte chemotactic proteins (MCPs) secreted by human phagocytes

ELSEVIER

JOURNALOF IMMUNOLOGICAL METHODS Journal of Immunological Methods 174 (1994) 237-247

Leukocyte recruitment by monocyte chemotactic proteins (MCPs) secreted by human phagocytes A. Wuyts, P. Proost, W. Put, J.-P. Lenaerts, L. Paemen, J. Van Damme * Laboratory of Molecular Immunology, Rega Institute, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium

Abstract Phagocyte recruitment is an important immunological phenomenon in inflammation and cancer. A large family of selective chemotactic cytokines, designated chemokines, has recently emerged. Interleukin-8 (IL-8) is the prototype of such neutrophil activating factors, whereas MCP-1 is a well studied monocyte chemotactic protein. In vitro chemotactic assays were used to isolate and identify natural chemokines from mononuclear phagocytes and tumor cells. Additional new chemotactic proteins (MCP-2, MCP-3) attracting monocytes were also discovered by these methods. All chemokines are structurally related and show affinity for heparin. MCP-1, -2 and -3 have a comparable specific activity in monocyte chemotaxis assays. Specific and sensitive radioimmunoassays for MCP-1 and IL-8 were developed to study the regulation of their secretion by leukocytes. Monocytes or monocyte tumor cells produce MCP-1 a n d / o r IL-8 in response to cytokines, virus, double stranded RNA, bacterial endotoxin, mitogen or phorbol ester. Granulocytes were found to secrete only minor amounts of MCP-1 and IL-8. Keywords: Chemotaxis; Chemokine; Monocyte chemotactic protein; Interleukin

1. Introduction Migration of phagocytes during the inflammat o r y r e s p o n s e is m e d i a t e d by a n u m b e r o f c h e m o -

tactic cytokines, d e s i g n a t e d c h e m o k i n e s . V a r i o u s cell t y p e s s e c r e t e m u l t i p l e c h e m o k i n e s a f t e r stimu l a t i o n with d i f f e r e n t i n d u c e r s . T h e h a l l m a r k o f t h e s e c h e m o k i n e s is t h e c o n s e r v a t i o n o f f o u r cys-

Abbreviations: BSA, bovine serum albumin; ConA, concanavalin A; CPG, controlled pore glass; EMEM, Eagle's minimum essential medium; FCS, foetal calf serum; tMLP, formylmethionylleucyiphenylalanine; FPLC, fast protein liquid chromatography; GCP, granulocyte chemotactic protein; HBSS, Hanks' balanced salt solution; HSA, human serum albumin; IL-8, interleukin-8; LPS, iipopolysaechafide; MCP, monocyte chemotactic protein; PABA, protein A bacterial adsorbent; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; poly rI : rC, polyriboinosinic.polyribocytidylic acid; rlFN-y, recombinant interferon-'), RP-HPLC, reverse phase high performance liquid chromatography; PTH, phenylthiohydantoin; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TCIDs0/ml , 50% tissue culture infectious dose; TFA, trifluoroacetic acid. * Corresponding author. 0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All fights reserved SSDI 0022-1759(94)00102-3

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teine residues that are important for the tertiary structure of these proteins. Chemokines can be divided into two subfamilies, depending on whether the first two cysteines are adjacent, the C-C chemokines, or separated by one amino acid, the C-X-C chemokines (Oppenheim et al., 1991; Schall et al., 1991; Van Damme, 1991). Monocyte chemotactic proteins predominantly belong to the C-C subfamily, whereas neutrophil chemotactic proteins belong to the C-X-C subfamily. New chemokines have been identified either by studies on differential gene expression in stimulated cells or by purification of induced proteins from cell supernatants. The former method allows the detection of novel gene products that could be classified in the chemokine family based on structural similarity. The latter method is based upon biological test systems and protein sequence analysis of the purified chemotactic factor. Distinct in vitro assays have been used to study chemokines, including the release of specific enzymes by activated phagocytes (Masure et al., 1991) and the selective migration of cells under agarose or through micropore filters (Van Damme and Conings, 1991). The specificity of these biological tests depends both on the purity of the test cells and the microscopic discrimination between the migrated cells and on the purity of the tested chemotactic factor. The availibility of pure natural, recombinant or synthetic chemokines is important for the production of specific polyclonal or monoclonal antibodies necessary to develop specific immunoassays. With such tests, the regulated production of each of these biologically and structurally related molecules can be studied. In our laboratory, several new chemotactic factors have been isolated from in vitro cultured cells and identified as chemokines by sequence analysis of the pure proteins. These include the C-X-C chemokines granulocyte chemotactic protein-1 or interleukin-8 (GCP-1/IL-8; Van Damme et al., 1988) and granulocyte chemotactic protein2 (GCP-2; Proost et al., 1993) as well as the C-C chemokines monocyte chemotactic protein-l, -2 and -3 (MCP-1, MCP-2 and MCP-3; Van Damme et al., 1989; Van Damme et al., 1992).

2. Materials and methods

2.1. Isolation of granulocytes and monocytes from human peripheral blood Heparinized peripheral blood from a single donor was mixed with I vol. of phosphate-buffered saline (PBS) and 1 vol. of hydroxyethyl starch (Plasmasteril; Fresenius, Bad Homburg, Germany) in a cylinder and placed for 30 min at 37°C to allow sedimentation of erythrocytes. The supernatant was centrifuged at 400 x g for 7.5 min. The pellet was washed and suspended in PBS. The cell suspension was loaded carefully on 3 vols. of Ficoll-sodium metrizoate (Lymphoprep; Nyegaerd, Oslo, Norway, density = 1.077 g/ml) and this was centrifuged at 400 × g for 30 min (without the brake on). The layer of mononuclear cells was removed, washed and suspended in Hanks' balanced salt solution, supplemented with 0.1% human serum albumin (HBSS + 0.5% HSA). Mononuclear cells were used as a source for monocytes. Double-distilled water was added to the cell pellet that contained granulocytes and remaining erythrocytes, to lyse the erythrocytes. After 30 s, NaC1 solution was added to make the cell suspension isotonic. The suspension was centrifuged at 250 x g for 7.5 min before the granulocyte pellet was washed and suspended in HBSS + 0.1% HSA. For the agarose migration assay, granulocytes and mononuclear cells were diluted in HBSS + 0.1% HSA to 3 x 107 and 108 cells per ml, respectively. For chemotaxis in the microchamber, cells were diluted to 106 cells per ml for granulocytes and to 2 x 10 6 for mononuclear ceils. The cells were immediately used in the assays.

2.2. Chemotaxis under agarose In the agarose migration assay (Nelson et al., 1975), the migration distance of cells under agarose towards test samples is used as a parameter for the chemotactic potency of the samples. Agarose plates were prepared by mixing the prewarmed (48°C) solution A, consisting of 7 ml foetal calf serum (FCS; Gibco, Paisley, Scotland),

A. Wuyts et al. /Journal of lmmunological Methods 174 (1994) 237-247

7 ml of 10 X concentrated Eagle's minimum essential medium (EMEM, Gibco), 1.4 ml of sodium bicarbonate 7.5%, 0.7 ml glutamine 200 mM and 20 ml pyrogen-free distilled water with solution B, consisting of 0.63 g agarose (Indubiose A 37 HAA, IBF, Villeneuve-la Garenne, France), boiled in 35 ml of distilled water until complete dissolution and subsequently cooled to 48°C. 6 ml of solution AB was poured into 6 cm (i.d.) plastic tissue culture dishes (Nunc, Denmark) and allowed to cool (30 min). Six series of three wells were made in the agarose-gel (3 mm i.d., 3 mm inter-space). The plates were incubated at 37°C in a 5% CO 2 incubator while the samples and cells were prepared. The centre well of each series of three wells was filled with 10 /zl of cell suspension (3 x 105 granulocytes or 106 mononuclear cells). The inner well was filled with 10/zl of non-chemotactic control medium (HBSS + 0.1% HSA) and the outer well was filled with 10/xl of sample dilution or 10 /xl of a positive control such as N-formylmethionylleucylphenylalanine,10-7 M (fMLP, Sigma, St. Louis, MO, USA), purified IL-8 (100 ng/ml) for granulocytes (Van Damme et al., 1988) or MCP-1 (1/zg/ml) for monocytes (Van Damme et al., 1989). The agarose plates were incubated at 37°C in a 5% CO2 incubator for 2 or 3 h for granulocytes and monocytes, respectively. The assay was stopped by adding 3 ml methanol to the agarose plates. The effective migration distance (migration distance towards the sample minus migration distance towards the negative control) was measured. The chemotactic potency of the chemokines was expressed in units, 1 U / m l corresponding to the half-maximal effective migration distance obtained with the corresponding positive control.

2.3. Chemotaxis through micropore filters In the microchamber migration assay (Falk et al., 1980), the number of cells that migrate actively through a filter with pores of a precise size towards test samples is used as a parameter for chemotactic potency of the samples. A 5/zm pore-size polyvinylpyrrolidone-free(for granulocytes) or polyvinylpyrrolidone-treated (for

239

monocytes) polycarbonate filter (Nuclepore, Pleasanton, CA, USA) was placed in a 48-well chemotaxis chamber (Neuro Probe, Cabin John, MD, USA), so that two compartments were created. The lower compartment was filled with 27/zl of sample dilutions, a positive control (fMLP 10 -s M, IL-8 for granulocytes (10 ng/ml) or MCP-1 (100 ng/ml) for monocytes) or a negative control (HBSS + 0.1% HSA), the upper compartment was filled with 50/zl of granulocytes (0.5 x 105 cells) or mononuclear cells (105 cells) in HBSS + 0.1% HSA. The chamber was incubated at 37°C in a 5% CO 2 incubator for 45 or 120 min for the granulocytes and monocytes respectively. The filter was removed from the chamber, the cells were fixed with 70% methanol and stained with DiffQuick (Harleco, Gibbstown, N J, USA). The cells which had migrated through the filter, were counted in ten microscopic fields per well. The chemotactic index, corresponding to the number of cells having migrated to the sample, divided by the number of cells having migrated to the negative control, was calculated. The chemotactic activity of the samples was expressed in units, 1 U / m l corresponding to a chemotactic index of 2.5. 2.4. Regulated production of chemokines in various

cell types Human osteosarcoma cells (MG-63) were grown in EMEM containing 10% FCS. Confluent monolayers (175 cm 2, Nunc, Roskilde, Denmark) were stimulated in EMEM (70 ml) containing 0.5% FCS for 48 h at 37°C with a crude cytokine mixture to produce chemokines on a large scale. Myelomonocytic THP-1 cells were grown in RPMI 1640 medium (Gibco) containing 10% FCS. They were seeded at 5 x 10 6 cells/ml in stationary cultures (25 cm2; Nunc) in RPMI 1640 medium (5 ml) containing 2% FCS for induction during 48 h at 37°C with measles virus (Attenuvax strain, 105.6 50% tissue culture infectious dose per ml (TCIDs0/ml)), the double-stranded RNA polyriboinosinic : polyribocytidilic acid (poly rI : rC; P-L Biochemicals, Milwaukee, WI, USA), rlFN-y (Boehringer Mannheim, Mannheim, Germany), purified natural human IL-1/3 (Van Damme et

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al., 1988), phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO, USA) and lipopolysaccharide (LPS; Escherichia coli 0.Ill.B4; Difco, Detroit, MI, USA). Human peripheral blood mononuclear cells were isolated on Ficoll-sodium metrizoate, as previously described. The mononuclear cell fraction was incubated in stationary cultures (25 cm 2) at 2.5 x 106 cells/ml in serum-free EMEM (5 ml) for 2 h at 37°C. Non-adherent cells were removed by repeated washes with serum-free medium and the cultures were replenished with EMEM. These monocyte cultures were stimulated in serum-free EMEM for 48 h at 37°C with different concentrations of measles virus, poly rI:rC, rlFN-y, natural human IL-1/3, the mitogen concanavalin A (ConA; Calbiochem, San Diego, CA, USA), LPS and PMA. Granulocytes were isolated from heparinized human peripheral blood as previously described. Cells were incubated at 2.5 x 106 cells per ml in stationary cultures (25 cm 2) in EMEM containing 0.5% serum. The cells were stimulated with different concentrations of rIFN-y, natural IL-1/3, LPS and PMA for 16 h at 37°C. 2.5. Purification o f new chemokines

Chemotactic activity in the supernatant (3000 ml) from MG-63 osteosarcoma cells was first concentrated and partially purified by adsorption to controlled pore glass (CPG-10-350; Serva, Heidelberg, Germany) at neutral pH and subsequent elution with 0.3 M glycine/HCl, pH 2.0 at 4°C (Van Damme et al., 1987). The concentrated eluate was dialysed against 50 mM Tris/HCI, 50 mM NaCI pH 7.4. The dialysed eluate of CPG was loaded on a heparin-Sepharose (CL-6B; Pharmacia, Uppsala, Sweden) column, equilibrated with 50 mM Tris/HC1, 50 mM NaCI pH 7.4 (10 ml/h). The column was washed with the equilibration buffer and proteins were eluted in a linear NaCI gradient (0.05-2 M) in 50 mM Tris/HCl, pH 7.4 (20 m l / h , 5 ml fractions). Protein concentrations were measured by a Coomassie blue G-250 binding assay (Bradford, 1976), using the Bio-Rad (Rich-

mond, CA, USA) commercial kit and bovine serum albumin (BSA) as a standard. Fractions containing the chemotactic activity eluting from the heparin-Sepharose column were dialysed against 50 mM formate, pH 4.0 and further purified by Mono S cation exchange fast protein liquid chromatography (FPLC; Pharmacia). The column, equilibrated with 50 mM formate pH 4.0, was loaded and washed with the equilibration buffer at 1 ml/min. The proteins were eluted in a linear NaCI gradient (0-1 M) in 50 mM formate, pH 4.0 (1 ml/min, 1 ml fractions). A280 was monitored as a parameter for protein concentration. Finally, FPLC fractions with chemotactic activity were purified by reverse phase high performance liquid chromatography (RP-HPLC). Samples were injected on a 220 x 2.1 mm C-8 Aquapore RP-300 column (Applied Biosystems Inc., Foster City, CA, USA), equilibrated with 0.1% trifluoroacetic acid (TFA) in water (solvent A) and the proteins were eluted with an acetonitrile gradient (solvent B, 80% acetonitrile, 0.1% TFA in water; 0.4 ml/min, 0.4 ml fractions). A 220 was monitored as a parameter for protein concentration. Fractions, derived from FPLC and HPLC were checked for purity by SDS-PAGE (Laemmli, 1970) under reducing conditions. Samples (3-20 ~1) were loaded onto a linear gradient (10-25%, w/v) polyacrylamide gel, containing 0.1% (w/v) SDS, with a 5% (w/v) stacking gel, containing 0.25% (w/v) SDS. The dimensions of the gel were 17 x 13 x 0.1 cm. The proteins were made visible by silver staining (Guevara et al., 1982). The relative molecular mass markers (Bio-Rad Laboratories, Richmond, CA, USA) phosphorylase b (M r 92500), BSA (M r 66200), ovalbumin (M r 45 000), carbonic anhydrase (M r 31000), soybean trypsin inhibitor (M r 21500) and lysozyme (M r 14400) and the low relative molecular mass marker (Pierce Chemical Co., Rockford, IL, USA) aprotinin (M r 6500) were used. 2.6. NH2-terminal amino acid sequence analysis

The amino acid sequence of proteins was determined by Edman degradation on a pulsed liq-

A. Wuytset al. /Journal of Immunological Methods 174 (1994) 237-247

uid (477A/120A) amino acid sequencer (Applied Biosystems) with on-line PTH-amino acid analysis. Cysteine residues were determined by on-filter reduction and modification with tributylphosphine and 4-vinylpyridine (Aldrich Chemical Co., WI, USA) (Andrews and Dixon, 1987). 2. 7. Radioirnmunoassays for IL-8 and MCP-1

Mononuclear cell-derived IL-8 was purified to homogeneity from the supernatant of these cells by adsorption to silicic acid, heparin-Sepharose chromatography and cation exchange FPLC (Van Damme et al., 1988). The purity of IL-8 was checked by SDS-PAGE and by NH2-terminal amino acid sequence analysis. Pure natural IL-8 supplemented with Freunds adjuvant (Difco, Detroit, MI, USA) was repeatedly injected into a goat to prepare a specific antiserum. 125I-IL-8 (2000 Ci/mmol) was purchased from Amersham (Buckinghamshire, UK). Recombinant or natural MCP-1 supplemented with Freund's adjuvant was injected in rabbits to prepare a specific antiserum. MCP-1 was labelled with N-succinimidyl 3-(4-hydroxy,5-[125I]iodophenyl)propionate (Amersham; Bolton and Hunter, 1973). 5 /xg of MCP-1 in 10 ~1 of 0.1 M borate buffer pH 8.5 were added to the dried iodinated ester. This mixture was agitated for 15 min at 0°C. Unchanged ester was reacted with 0.5 ml of 0.2 M glycine in 0.1 M borate buffer pH 8.5 for 5 min at 0°C to obviate subsequent conjugation to carrier proteins. The 125I-labelled protein was separated from the other labelled products of the conjugation reaction, glycine conjugate and 3-(4hydroxyphenyl)propionic acid by gel filtration. Samples, IL-8 standard and MCP-1 standard were diluted in Tris-buffered saline pH 7.4 (TBS), containing 11% polyethylene glycol 6000, 0.5% protamine sulfate (Sigma), 10 mM EDTA and 0.1% sodium azide. 125I-IL-8, 125I-MCP-1, antiserum and protein A bacterial adsorbent (PABA, Sigma) were diluted in TBS, containing 10 mM EDTA, 0.1% sodium azide and 1.0% BSA and 0.2% gelatin to ensure low non-specific binding (TBS-gel). To measure the amount of IL-8 or MCP-1 produced by stimulated cells, 100 /.d aliquots of

241

dilutions of the supernatant, the IL-8 standard or MCP-1 standard were mixed with 50/zl anti-IL-8 antiserum (diluted 1/3000) or anti-MCP-1 antiserum (diluted 1/3000) and 50 izl 125I-IL-8 (100 pg) or 125I-MCP-1 (100 pg), respectively. After 18 h incubation at room temperature, 50/~1 of PABA 2.5% were added to precipitate antibody-bound reactivity. 1 ml TBS-gel was added after 30 min, followed by centrifugation (5000 × g , 10 min). Supernatants were removed and antibody-bound reactivity was counted in the pellets in a PRIAS Auto-gamma Packard counter (Parkland, Warrenville, IL, USA) during 1 min. The concentrations of IL-8 and MCP-1 in the samples were determined from the standard curves.

3. Results and discussion

3.1. Isolation and in vitro activity o f specific monocyte and neutrophil chemotactic cytokines

MG-63 osteosarcoma cells were stimulated with a crude cytokine mixture to produce chemokines. The supernatant was concentrated and purified by adsorption to CPG beads, followed by heparin-Sepharose chromatography. The proteins, eluted from the heparin-Sepharose column, were tested for chemotactic activity for neutrophils and monocytes in the agarose migration assay. Since neutrophil and monocyte chemotactic activities showed similar affinity for heparin, this method did not permit their separation (Fig. 1). The heparin-Sepharose fractions with chemotactic activity were pooled and further purified by cation exchange FPLC. Neutrophil and monocyte chemotactic activity could be separated by this purification step indicating that the biological activity was mediated by different factors. The main peak of neutrophil chemotactic activity eluted at 0.95 M NaCI in the gradient and corresponded to a single protein band of 6-7 kDa upon SDS-PAGE. This protein was identified as IL-8 by NH2-terminal sequence analysis (Van Damme et al., 1988). Recently, a new granulocyte chemotactic protein (GCP-2) has also been isolated by FPLC using the microchamber assay (Proost et al., 1993).

A. Wuyts et al. /Journal of Immunological Methods 174 (1994) 237-247

242

!+

3000

2

50

40

/ J/////J"

o

i

T

-

30

E

20 o

E 5

10

0

0 5

10

15

20

25

30

fraction number

Fig. 1. Heparin-Sepharose chromatography of monocyte and granulocyte chemotactic activity. Concentrated and partially purified cell supernatant from MG-63 osteosarcoma ceils (stimulated with a crude cytokine mixture) was loaded on a heparin-Sepharose column at pH 7.4 and eluted (5 ml fractions) with a linear NaCI gradient ( - - - - --). Protein concentrations were determined by a Coomassie blue G-250 binding assay (,x). Fractions were tested for neutrophil and monocyte chemotactic activity in the agarose migration assay (histograms).

The major peak of monocyte chemotactic activity eluted on FPLC at 0.5 M NaCI in the gradient. This monocyte chemotactic protein was identified as MCP-1 (Van Damme et al., 1989). The sequence was found to be identical to that obtained by others (Furutani et al., 1989; Yoshimura et al., 1989a). Additional peaks of monocyte chemotactic activity eluting at higher NaCI molarity, designated MCP-2 and MCP-3, were further purified in parallel with MCP-1 by RP-HPLC. MCP-1, MCP-2 and MCP-3 eluted on HPLC at 26%, 30% and 27.5% acetonitrile respectively

(Van Damme et al., 1992). MCP-1 was heterogeneous on SDS-PAGE, in that multiple molecular mass bands (10, 12, 14 and 16 kDa) were found in single HPLC fractions. MCP-2 and MCP-3 corresponded to protein bands migrating at 7.5 and 11 kDa, respectively. Sequence analysis of the proteins confirmed that MCP-1, MCP-2 and MCP-3 are structurally related chemokines (Fig. 2; Van Damme et al., 1992). In the agarose migration assay, IL-8 showed a specific activity of 105 U / m g for neutrophils, but GCP-2 had no chemotactic activity. In this assay,

CHEMOKINE

(% Identity)

MCP-I

QPDAINAPVTCCYNFTNP.KISVQELASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT

(100)

MCP-2

QPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAV~FKTERGKEVCADPKERWVRDSMKHLDQIFQNLKP

(62)

MCP-3

QPVGINTSTTCCYRFINKKIPKQRLESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL

(71)

RANTES

SPYSSDTTPCCFAYIARPLPRAHIKEY

FYTSGKCSNPAWFVTRKNRQVCANPEFd~WVREYINSLEMS

(28)

MIP-I~

SLAADTPTACCFSYTSRQIPQNFIADY

FETSSQCSKPGVIFLTKRSRQVCADPSEEWVQKYVSDLELSA

(38)

MIP-IK

APMGSDPPTACCFSYTARELPRNFVVDY

YETSSLCSQPAWFQTKRSKQVCADPSESWVQEYVYDLELN

(36)

1-309

SKSMQVPFSRCCFSFAEQEIPLRAILCY

P.NTSSICSNEGL~FKLKRGKEACALDTVGWVQRHREMLP.HCPSKP.K

(31)

Fig. 2. Amino acid sequence similarity of human C-C chemokines. Protein sequences of MCP-2 (Chang et al., 1989b; Van Damme et al., 1992), MCP-3 (Van Damme et al, 1992; Opdenakker et al., 1993), RANTES (Schall et al., 1988), MIP-la (Zip~l et al., 1989), MIP-lfl (Chang and Reinhe~, 1989) and 1-309 (Miller et al., 1989) were compared with the sequence of MCP-1 (Furutani et al., 1989; Yoshimura et al., 1989).

A. Wuytset al. /Journal of Immunological Methods 174 (1994) 237-247 MCP-2 and MCP-3 had a specific activity for monocytes of about 104 U / m g , comparable with that of MCP-1. The biological effect of G C P s and MCPs was also tested in the microchamber migration assay. IL-8 showed chemotactic activity for neutrophils with a specific activity of > 2 / 1 0 6 U / m g , whereas that of GCP-2 was a ten-fold lower. MCPs and fMLP showed a bell-shaped dose-response curve for chemotactic activity on monocytes, typical for chemotactic factors. MCP-1, MCP-2 and MCP-3 had specific activities of about 8 X 105 U / m g , 3 x 105 U / m g and 2 x l0 s U / m g , respectively (Table 1). This indicates that the microchamber assay is at least ten-fold more sensitive than the agarose assay. However, the latter assay is easier to perform when large series of fractions need to be tested. In both assay systems IL-8 and GCP-2 did not attract monocytes whereas MCPs did not stimulate neutrophil chemotaxis.

3.2. Induction o f MCP-1 and IL-8 in macrophages Monocytes and granulocytes were stimulated to produce chemokines with different inducers at different concentrations. IL-8 and MCP-1, released by the stimulated cells, were measured by a specific R I A using pure 125I-chemokines (Table 2; R a m p a r t et al., 1992). Myelomonocytic THP-1 cells produced IL-8 and MCP-1 after stimulation with LPS (0.5 / z g / m l ) and P M A (10 n g / m l ) . MCP-1, but not IL-8, was produced in response to IFN-7 (20 n g / m l ) and measles virus (104.3 T C I D s 0 / m l ) . ILlfl and poly rI : rC were completely ineffective on THP-1 cells. A d h e r e n t mononuclear cells produced significant levels of both IL-8 and MCP-1 in response to IL-1/3 (100 U / m l ) , measles virus (10 4.3 T C I D s 0 / m l ) , and C o n A (10 /zg/ml). P M A and LPS predominantly stimulated the release of IL-8 (but not MCP-1), whereas IFN-7 and poly r I : r C were only marginal inducers of MCP-1 on monocytes. Granulocytes did not produce IL-8, nor MCP-1 after stimulation with IL-1/3, I F N - 7 or PMA, but LPS showed a weak induction of IL-8.

Table 1 Chemotactic activity of MCPs Concen- Chemo- U/ml tration tactic index MCP-I 100 ng/ml 4.6 2600 U/ml 3/~g/ml 10 ng/ml 7.2 1 ng/ml 2.2 0.1 ng/ml 2.6 MCP-2 100 ng/ml 1.2 340 U/ml 1/zg/ml 10 ng/ml 3.9 1 ng/ml 1.3 0.1 ng/ml 0.5 MCP-3 100 ng/ml 0.2 200 U/ml 1/.tg/ml 10 ng/ml 2.9 1 ng/ml 1.6 0.1 ng/ml 0.5 fMLP 10 7 M 1.7 fMLP 10 s M 6.1

243

Specific activity 8 x 105 U/mg

3 x 105 U/mg

2 x 105 U/mg

MCP-1, MCP-2 and MCP-3 were tested in the microchamber migration assay for monocyte chemotactic activity. The chemotactic index corresponds to the number of cells migrated towards the sample, divided by the number of cells migrated towards the negative control; 1 U/ml corresponds to a chemotactic index of 2.5. fMLP was used as a positive control.

Monocytes and THP-1 cells could secrete IL-8 and MCP-1, whereas granulocytes only produced IL-8. A single stimulus (e.g., viral infection) can stimulate the release of both IL-8 and MCP-1. The production of IL-8 and MCP-1 is, in part, cell- or inducer-specific. In addition, there is specificity in that IL-8 is active on neutrophils but not on monocytes, whereas MCP-1 is chemotactic for monocytes but not for neutrophils.

3.3. Monocyte chemotactic factors of the chemokine family The chemotactic cytokines with four conserved cysteine residues can be divided into two subclasses: the C-X-C subclass, that contains chemokines of which the two NH2-terminal cysteines are separated by one amino acid, and the C-C subclass, that contains chemokines of which the two NH2-terminal cysteines are adjacent. Neutrophil chemotactic factors (Van D a m m e , 1991), e.g., IL-8, G R O , NAP-2, IP-10, as well as the recently identified ENA-78 (Walz et al., 1991)

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A. Wuyts et al. /Journal of lmmunological Methods 174 (1994) 237-247

and GCP-2 (Proost et al., 1993) are C-X-C chemokines, whereas monocyte chemotactic factors (Oppenheim et al., 1991; Schall, 1991), e.g., MCP-1, MCP-2, MCP-3, RANTES, M I P - l a , MIP-1/3 and 1-309 are C-C chemokines. The sequence similarity of the C-C chemokines is shown in Fig. 2. MCP-1 is produced by mononuclear leukocytes (Yoshimura et al., 1989b; Van Damme et al., 1989; Decock et al., 1990), fibroblasts (Larsen et al., 1989; Strieter et al., 1989; Van Damme et al., 1989);, endothelial cells (Strieter et al., 1989; Sica et al., 1990), smooth muscle cells (Yu et al., 1992), epithelial cells (Elner et al., 1991; Paine et al., 1993), melanocytes (Zachariae et al., 1991), keratinocytes (Barker et al., 1991), chondrocytes (ViUiger et al., 1992), osteoblastic cells (Williams et al., 1992), mesothelial cells (Jonjic et al., 1992), mesangial cells (Brown et al., 1992) and tumor cells (Graves et al., 1989; Matsushima et al., 1989; Van Damme et al., 1989, 1992; Yoshimura et al., 1989c; Zachariae et al., 1990; Kasahara et al., 1991; Standiford et al., 1991). MCP-1 is a chemoattractant for monocytes (Van Damme et al., 1989; Leonard and Yoshimura, 1990) and induces

histamine release in basophils (Alam et al., 1992a). MCP-2 and MCP-3 are secreted by osteosarcoma cells (Van Damme et al., 1992) and monocytes (Chang et al., 1989a; Minty et al., 1993) and are chemotactic for monocytes, but not for neutrophils (Van Damme et al., 1992); other cell sources are at present under investigation. RANTES is produced by leukocytes and tumor cells (Schall, 1991) and attracts monocytes and a subset of memory T cells (Schall et al., 1990). R A N T E S is also chemotactic for eosinophils (Kameyoshi et al., 1992; Alam et al., 1993) and induces histamine release in basophils (Kuna et al., 1992). M I P - I a is produced by fibroblasts, leukocytes and tumor cells (Schall, 1991); it is a chemoattractant for monocytes (Wang et al., 1993), activated CD8 ÷ T cells (Taub et al., 1993), eosinophils (Rot et al., 1992) and basophils (Alam et al., 1992b). MIP-1/3 is produced by leukocytes and tumor cells (Schall, 1991) and shows chemotactic activity for monocytes (Wang et al., 1993) and activated CD4 ÷ T cells (Taub et al., 1993).

Table 2 Production of MCP-1 and IL-8 by stimulated phagocytes Inducer

Measles virus (TCIDs0 1056) Poly rI : rC rlFN--y IL-1/3 ConA LPS

PMA Co

THP-1 cells

1/20 1/200 100 p . g / m l 10/xg/ml 200 n g / m l 20 n g / m l 100 U / m l 10 U / m l 10/~g/ml 1 ~g/ml 50/zg/ml 5 ~g/ml 0.5 ~ g / m l 100 n g / m l 10 n g / m l

Monocytes

Granulocytes

IL-8

MCP-1

IL-8

MCP-1

IL-8

MCP-I

3 1 3 2 4 2 2 2 ND ND 160 80 14 > 200 > 200 2

32 2 2 1 158 210 1 1 ND ND 195 169 72 16 20 1

140 41 17 12 9 8 88 28 220 37 240 220 ND 30 54 12

36 9 10 4 7 5 12 10 28 24 6 8 ND 0 5 2

ND ND ND ND 6 ND 21 4 ND ND 24 34 ND 3 2 19

ND ND ND ND 1 ND 4 < 1 ND ND < 1 < 1 ND < 1 1 1

Myelomonocytic THP-1 cells, freshly isolated monocytes and granulocytes were stimulated with measles virus, poly r I : r C , recombinant IFN-3,, natural IL-1/3, ConA, LPS and P M A at different concentrations. IL-8 and MCP-1, released by the stimulated cells, were m e a s u r e d in the supernatant by a specific R I A for IL-8 and MCP-1, respectively. Concentrations of IL-8 and MCP-1 are expressed in n g / m l . N D = not determined.

A. Wuyts et al. /Journal of lmmunological Methods 174 (1994) 237-247

1-309 is produced by activated T lymphocytes and is a chemoattractant for monocytes (Miller and Krangel, 1992). The biological effects of chemokines observed in vitro could also be confirmed in vivo. Intradermal injection of IL-8 resuited in selective neutrophil emigration and oedeme formation, whereas MCP's predominantly attracted monocytes (Rampart et al., 1992; Van Damme et al., 1992). The possible role of chemokines in pathology must therefore be envisaged in detail. Immunoassays and bioassays are at present used to investigate chemokines in body fluids.

Acknowledgements This work was supported by the National Fund for Scientific Research (NFWO). A. Wuyts is a research assistent of the NFWO.

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