JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 20:615–621 (2000)Mary Ann Liebert, Inc.

Selective Induction of the Monocyte-Attracting ChemokinesMCP-1 and IP-10 in Vesicular Stomatitis Virus-Infected

Human Monocytes

DELIA BUßFELD,1 MARIANNE NAIN,1 PETER HOFMANN,1 DIETHARD GEMSA,1

and HANS SPRENGER2

ABSTRACT

It is characteristic of viral infections that monocytes/macrophages and lymphocytes infiltrate infected tissue,and neutrophils are absent. CC and non-ELR CXC chemokines predominantly attract mononuclear leuko-cytes, whereas the ELR motif-expressing CXC chemokines primarily act on neutrophils. To investigate thegeneral role of chemokines in viral diseases, we determined their release and expression patterns after infec-tion of human monocytes with vesicular stomatitis virus (VSV). Human monocytes were productively infectedby VSV. Surprisingly, VSV did not induce the release of the proinflammatory cytokines tumor necrosis fac-tor-a (TNF-a), interleukin-1b (IL-1b ), and IL-6. In contrast, we found a strong induction of the CC chemokinemonocyte chemotactic protein-1 (MCP-1) and the non-ELR CXC chemokine interferon-c (IFN-c ) inducibleprotein-10 (IP-10) by VSV on the gene and protein level. The expression and release of the neutrophil chemoat-tractants IL-8 and growth-related oncogene-a (GRO-a) remained unaffected after VSV infection. Our resultsindicate that the typical monocyte and lymphocyte-dominated leukocyte infiltration of virus-infected tissue isbased on a selective induction of mononuclear leukocyte-attracting chemokines.

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INTRODUCTION

T ISSUE INFLAM M ATIO N IS CHARACTER IZED by the recruitment,immigration, and activation of leukocytes. Chemoattractant

cytokines (chemokines) play a crucial role in transendothelialdiapedesis of leukocytes and their movement through the extra-cellular matrix.(1–3) Most viral diseases are accompanied by aspecific infiltration dominated by mononuclear leukocytes, andneutrophils are absent as long as no bacterial superinfection oc-curs. Numerous viral infections of macrophages or monocyteshave been shown to induce the expression of such cytokines astumor necrosis factor- a (TNF-a ), interleukin-1 (IL-1), and IL-6 by influenza A or Coxsackie B3 virus,(4–7) whereas much lessis known about virus-induced chemokine release.

Chemokines are immunoregula tory proteins that attract dis-tinct leukocyte subpopulations. (8,9) The main structural charac-teristics of chemokines are four conserved cysteines. Depend-ing on their position, the chemokine family can be subdividedinto CXC, CC, C, and CX3C branches. (3,10) Members of the CCchemokine subfamily, such as macrophage inflammatory pro-tein-1 a (MIP-1 a ) and monocyte chemotactic protein-1 (MCP-

1), preferentially attract monocytes and lymphocytes. (1 1) TheCXC chemokines lacking an ELR motif amino-termina l of thefirst cysteine, interferon- g (IFN- g ) inducible protein-10 (IP-10),act on the same target cells. In contrast, CXC chemokines withan ELR domain, such as IL-8 and growth-related oncogene- a(GRO- a ), are major neutrophil chemoattractants .

We were able to elucidate the mechanisms regulating the se-lective immigration of mononuclear leukocytes by influenza Avirus. After infection of human monocytes increased expres-sion of the mononuclear cell (MNC)-attracting CC chemokinesMIP-1 a , MCP-1, and RANTES occurred, whereas expressionof the prototype neutrophil CXC chemoattractants IL-8 andGRO- a was not affected or even suppressed. (12) Based on thesefindings, we speculated that the influenza virus-dependent in-duction of only MNC-attracting chemokines may be responsi-ble for the preferential influx of mononuclear leukocytes intovirus-infected tissue.

To examine whether this selective induction of MNC-at-tracting chemokines by influenza A virus may represent a gen-eral feature of virus-specific immune responses, we infected hu-man monocytes with another prototype RNA virus, vesicular

1Institute of Immunology, Philipps University, Marburg, Germany.2Institute of Laboratory Medicine, Leopoldina-Hos pital, Schweinfurt, Germany.

stomatitis virus (VSV), and analyzed the responding chemokinepattern. We report the selective induction of the MNC-attract-ing chemokines MCP-1 and IP-10 in human monocytes afterVSV infection. Expression of the neutrophil-attracti ng chemo-kines IL-8 and GRO- a remained unaffected.

MATERIALS AND METHODS

Cell preparation and culture

Human monocytes were prepared from the buffy coat ofhealthy donors (generously provided by the Departm ent ofTransfusion Medicine, University of Marburg, Germany). Af-ter separation of the MNC by Ficoll-Hypaqu e density gradientcentrifugation, (4) the monocytes were enriched by elutriation toa purity of . 90% as determ ined by nonspecific esterase stain-ing or FACS® analysis using FITC-labeled anti-CD14 (Im-munotech, Hamburg, Germany).

Virus preparation and infection of monocytes

VSV, strain Indiana, was propagated on confluent My-coplasma -free HeLa cells, which were infected with 0.1 multi-plicity of infection (moi) VSV under serum-free conditions. Af-ter adsorption for 1 h, the supernatant was replaced by newmedium, and the cells were incubated until a cytopathic effectbecame clearly evident. The supernatant was collected, and thecells were quickly frozen and thawed three times. After cen-trifugation at 1000g and 4°C, the VSV-containing supernatantwas aliquoted and stored at 2 70°C. Infectivity was assessed bya standard plaque assay as cytopathic effect on confluent culturesof Mycoplasma-free HeLa cells with a 0.5% agarose overlay. (13)

Human monocytes (0.5 3 106/0.2 ml) were infected ormock-infected by exposure to VSV in serum-free minimum es-sential medium (MEM). After adsorption for 1 h, the virus-con-taining medium was replaced by 1 ml fresh, virus-free MEM.Lipopolysacchar ide (LPS) (10 ng/ml) from Escherichia colistrain 0127:B8 (Difco, Detroit, MI) was used as a positive con-trol for cytokine induction. Culture supernatants were collectedat various times after infection and stored in aliquots at 2 70°C.The remaining cells were used for RNA preparation.

Determination of cytokines

Chemokine levels were determined by sandwich enzyme-linked immunosorben t assays (ELISA) developed in our labo-ratory and previously described. (14) The IL-6 ELISA was per-formed with a monoclonal antibody (mAb) in combination witha polyclonal biotin-labeled antibody (both from Pharmingen,Hamburg, Germany) and streptavidin-perox idase (BoehringerMannheim, Mannheim, Germany). Release of TNF- a was de-termined by a highly specific ELISA that was established withmonoclonal antihuman TNF- a antibodies (kindly provided byBASF/Knoll AG, Ludwigshafen, Germany). (4)

The levels of IFN type I in culture supernatants were deter-mined by inhibition of the cytopathic effect of VSV on humanHeLa cells as previously described. (15) The IFN titer was cal-culated by comparison with a calibration curve established withrecombinant human IFN-a (rHuIFN- a ) (provided by Ernst-Boehringer Institute, Vienna, Austria).

Viral protein synthesis

Newly synthesized viral proteins in human monocytes (3 3106/3 ml) were determined by a [35S]-methionine pulse for ei-ther 12 h or 24 h after infection, followed by immune precipi-tation, SDS-PAGE separation, and autoradiography as outlinedpreviously in detail. (4)

Generation and labeling of riboprobes

Probes 300–400-bp long corresponding to human TNF-a ,MCP-1, MIP-1 a , IP-10, IL-8, and GRO-a were generated byRT-PCR and subsequent cloning of the respective PCR products.Total RNA (1 m g) from LPS-stimulated human monocytes wasoligo-dT-primed and reverse-transcribed with Superscript II re-verse transcriptase (Life Technologies, Eggenstein, Germany).The cDNA was amplified by specific forward and reverse primerscontaining artificial restriction sites at their 5 9 -ends by SuperTaqDNA polymerase (Stehelin, Basel, Switzerland). The amplifiedDNA was gel purified, digested with BamHI and EcoRI, and site-directed cloned into the respective sites of pBluescript SK (Strat-agene, LaJolla, CA). The digoxigenin (DIG)-labeled glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) antisense probe waskindly provided by Dr. A. Friedetzky (Institute of Immunology,University of Marburg, Germany). The specificity of all ribo-probes was confirmed by sequencing. Labeling efficiency wasexamined by dot blot analysis.

BUßFELD ET AL.616

FIG. 1. VSV protein synthesis in human monocytes. Humanmonocytes (3 3 106/3 ml) were left untreated (control) or ex-posed to VSV (2 moi) for 1 h, washed several times, resus-pended in fresh medium, and then pulsed with [35S]-methion-ine for 12 h or 24 h. After immunoprecipitatio n, all five VSVproteins are indicated in the autoradiogram: L, large protein; G,glycoprotein; N, nucleocapsid protein; P, phosphoprotein; M,matrix protein. Mr, molecular mass standards.

RNA preparation and Northern blot analysis

Total RNA was isolated by a modified guanidine thiocyanatemethod as reported in detail previously. (16) The membraneswere hybridized with DIG-labeled antisense riboprobesovernight under highly stringent conditions in 50% formamideat 68°C. Bound DIG-labeled riboprobes were nonradioactivelyvisualized using the DIG nucleic acid detection kit (BoehringerMannheim) and CDP-Star chemilumines cence substrate(Tropix, Bedford, MA, distributed by Boehringer Ingelheim ,Heidelberg, Germany).

Cycloheximide (CHX) was used to determine if the induciblechemokine gene expression was dependent on de novo proteinsynthesis. Monocytes (107) in 10 ml culture medium were stim-ulated with LPS (10 ng/ml) or exposed to VSV (2 moi) in thepresence or absence of 10 m g/ml CHX for 8 h. Thereafter, to-tal RNA was prepared and analyzed by Northern blot.

RESULTS

VSV replication in human monocytes and HeLa cells

At 8 h after infection of monocytes and HeLa cells with VSV,both cell types released infective virus particles into the super-natant. The yield of new viruses was much higher in HeLa cells(80 plaque-forming units [pfu]/0.5 3 106 cells) than in humanmonocytes (0.3 pfu). To exclude VSV-induced suppression ofmetabolism and effects on viability, the conversion of (3-[4,5-dimethylthiazol-2-yl]-2 ,5-diphenyltetrazolium bromide [MTT])

and release of lactate dehydrogenase (LDH) were determinedup to 20 h after exposure of human monocytes to LPS (10ng/ml) or VSV (2 moi). After this time, the metabolic activityof VSV-infected cells declined to , 50% of the untreated con-trol, but the release of LDH as a marker for cell lysis did notchange significantly (data not shown).

To examine the effects of VSV infection on human mono-cytes in more detail, viral protein synthesis was determined.Human monocytes were infected with 2 moi of VSV. After 12h and 24 h, newly synthesized proteins were marked with [35S]-methionine for 2 h. Electrophoretic separation of the proteinsrevealed that VSV dramatically inhibited the cellular proteinsynthesis. This inhibition was clearly evident (Fig. 1, 3rd lane)at 12 h after infection. In addition to viral proteins, only a fewcellular proteins were synthesized when compared with the non-infected control (Fig. 1, 2nd lane). After 24 h, VSV had com-pletely inhibited cellular protein synthesis and only viral pro-teins were detectable (Fig. 1, 4th lane). All five viral proteinswere found: large protein (L), glycoprotein (G), nucleocapsidprotein (N), phosphoprotein (P), and matrix protein (M).

Release of IFN- a / b and proinflam matory cytokinesfrom VSV-infected human monocytes

It was of particular interest whether monocytes would be ableto respond to viral infection with the release of cytokines de-spite the observed strong inhibition of protein synthesis in gen-eral. Human monocytes were exposed to LPS (10 ng/ml) or in-fected with VSV (2 moi). As expected, VSV infection inducedconsiderable IFN- a / b (IFN type I) activity when compared with

VSV-INDUCED CHEMOKINES 617

FIG. 2. Time course of MCP-1 (A) and IP-10 (B) release in VSV-infected human monocytes. Human monocytes (0.5 3 106/ml)were left untreated (open circles), exposed to 10 ng/ml LPS (closed circles), or infected with VSV (triangles) (2 moi). Chemokinerelease was determined by specific ELISA at the indicated times. Mean 6 SD of three experiments is shown.

untreated control cells. In contrast, VSV was not able to inducethe release of TNF- a or IL-1 b and IL-6 (data not shown).

Chemokine release after VSV infection

To compare the influenza A virus-induced chemokine pro-file of human monocytes (12) with that of VSV-infected cells,we examined the release of various chemokines. VSV selec-tively stimulated the release of the two monocyte-attrac tingchemokines MCP-1 and IP-10, which lasted for at least 20 h(Fig. 2). Whereas stimulation of monocytes with LPS led to astrong release of MCP-1 (Fig. 2A), it failed to induce IP-10(Fig. 2B). In addition, VSV failed to release the neutrophil-at-tracting chemokines IL-8 and GRO- a (Fig. 3).

Human monocytes responded to VSV exposure in a dose-dependent manner when they were infected with VSV (0.125–4moi). The release of MCP-1 and IP-10 had increased dose de-pendently (Fig. 4) 8 h after infection, with peak levels using1.5–2 moi.

Differential chemokine gene expression inVSV-infected human monocytes

In an attempt to explore the underlying molecular mecha-nisms, we studied chemokine gene expression by Northern blotanalysis after VSV infection. In accordance with the chemokineprotein data, we found an inducible MCP-1 and IP-10 mRNAaccumulation in human monocytes after VSV infection,whereas gene expression of the prototype proinflamm atory cy-tokine TNF-a and of the neutrophil-attracting chemokines IL-8

BUßFELD ET AL.618

FIG. 3. Release of neutrophil-attracti ng chemokines in VSV-infected human monocytes. The experiments were performedas described in Figure 2. After 20 h, the levels of IL-8 andGRO- a were determined by specific ELISA. Mean 6 SD ofthree identically prepared cultures.

FIG. 4. Dose-dependent release of chemokines after infection of human monocytes with VSV. Human monocytes (0.5 3 106/ml)were infected with the indicated doses of VSV (0.125–4 moi). After 8 h, the release of MCP-1 and IP-10 was determined by spe-cific ELISA. Mean 6 SD of three identically prepared cultures.

and GRO-a was not stimulated (Fig. 5). LPS strongly inducedthe expression of all analyzed chemokines except IP-10. Hy-bridization with sense riboprobes did not reveal specific signals(data not shown).

Dependence of VSV-induced IP-10 and MCP-1expression on de novo protein synthesis

To determine whether VSV directly stimulated the expres-sion of the two chemokines MCP-1 and IP-10 or it was de-pendent on de novo protein synthesis, we analyzed mRNA ex-pression in the presence of CHX. Human monocytes were leftuntreated, stimulated with LPS (10 ng/ml), or infected withVSV (2 moi) with or without CHX (10 m g/ml) for 8 h. CHXas an inhibitor of de novo protein synthesis did not influencethe low constitutive MCP-1 mRNA expression but entirelyabolished its induction by LPS or VSV (Fig. 6). A strikinglydifferent pattern was found for IP-10. CHX increased themRNA levels of IP-10 in unstimulated control cells but also inLPS-exposed and VSV-exposed monocytes, which indicates itsindependence of de novo protein synthesis.

DISCUSSION

The infiltration of monocytes and lymphocytes into infectedtissue and the absence of neutrophils are typical for viral in-

fections. We showed previously that infection of human mono-cytes by influenza A virus selectively increased the expressionof the MNC-attracting CC chemokines MIP-1 a , MCP-1, andRANTES, whereas the neutrophil-attracti ng CXC chemokinesIL-8 and GRO- a were not induced. (12) To explore the possiblegeneral importance of this selective induction of MNC-attract-ing chemokines during viral infections, we investigated themoderate response to another prototype-negativ e sense RNAvirus, VSV, which is a widespread virus that can also infect hu-mans. VSV infections display influenza-like symptoms, suchas fever, shivering, headache, retroorbital pain, myalgia,pharyngitis, vomiting, and diarrhea. Approximately 25% of allinfected patients develop vesicular lesions in the area of themouth, lips, or nose.(17)

Monocytes and macrophages are host cells for VSV repli-cation, (18) although lymphocytes have to be stimulated to in-crease their susceptibility to the virus. (19,20) The resistance offreshly isolated, resting mouse peritoneal macrophages to VSVwas abolished by injection of antibodies to mouse IFN-a / b (21,22) or was lost during in vitro cultivation of themacrophages, in parallel with decreasing IFN release. (23,24 )

The data presented here clearly show that human monocytesare indeed susceptible to infection by VSV. This is documentedby three lines of evidence: (1) the dramatic decrease of cellu-lar metabolism, (2) the de novo viral protein synthesis (Fig. 1),and (3) the induction of cytokines by VSV (Figs. 2–5). The ac-tual release of newly formed VSV from human monocytes wasrather low when compared with HeLa cells, which contradictsprevious observations (18) but supports the general concept thatleukocytes are not particularly permissive for viral replica-tion. (25) Nevertheless, a strong de novo protein synthesis of VSVproteins occurred in monocytes, and 24 h after infection, thevirus completely abolished the translation of cellular proteins(Fig. 1). This confirms the well-known observation that VSVis very effective in inhibiting both the transcription and the

VSV-INDUCED CHEMOKINES 619

FIG. 5. Selective expression of monocyte-attracting chemo-kines after VSV infection of human monocytes. Human mono-cytes (107/10 ml) remained either untreated (Con) or were ex-posed to 10 ng/ml LPS or 2 moi VSV. After 8 h, 2 m g of totalRNA was analyzed for TNF-a , MCP-1, IP-10, IL-8, GRO-a ,and GAPDH gene expression by DIG-labeled riboprobes. Arepresentative Northern blot analysis out of five is shown.

FIG. 6. Induction of IP-10, but not of MCP-1 gene expres-sion, by VSV is independent of de novo protein synthesis. Hu-man monocytes (107/10 ml) were either left untreated, stimu-lated with LPS (10 ng/ml), or infected with VSV (2 moi) for 8h in the presence or absence of 10 m g/ml CHX. Total RNA (2m g) was analyzed for MCP-1, IP-10, and GAPDH expressionby DIG-labeled riboprobes. A representative analysis out ofthree is shown.

translation of cellular proteins. (26 ,27) The viral M-protein seemsto play a key role in mediating this inhibition. (28,29) With in-cubation time beyond 20 h, HeLa cells(30) and human mono-cytes (31 ) finally became apoptotic.

We previously showed that infection of human monocyteswith influenza A virus or Coxsackie B3 virus induced TNF- a ,IL-1, and IL-6.(4 –6) In striking contrast, VSV was unable to in-duce any of these prototype proinflammato ry cytokines. It ap-pears, therefore, that VSV managed to evade the first proin-flammatory defense mechanisms mediated by the immedi-ate-early cytokines. As expected after most viral infections,VSV induced the release of IFN type I, which is consistent withpreviously reported data.(32)

Completely unexpected was the following novel observation.In striking contrast to the deficient proinflammato ry responseand in spite of the strong viral inhibition of the host cell me-tabolism, transcription, and protein synthesis, MCP-1 and IP-10 seem to escape this strong downregulation (Figs. 2 and 5).Both proteins preferentially attract monocytes and lymphocytesbut not neutrophils. (8,11) A viral origin of chemotactic activitycan be excluded, as neither VSV itself nor supernatants of VSV-exposed HeLa cells (mock-control) were chemotactic to mono-cytes. Neutrophil-attra cting chemokines were not induced(Figs. 3 and 5). We and others confirm ed MCP-1 as one of themost important proteins for host defense against viruses. Alongthis line, a strongly increased MCP-1 level was found in thecerebrospinal fluid (CSF) of AIDS patients with cy-tomegalovirus (CMV) encephalitis compared with AIDS pa-tients without viral infection, whereas MIP-1 a , MIP-1 b ,RANTES, and IL-8 were not induced. (33) Rösler et al.(34) pro-vided evidence that MCP-1 was the predominant chemokine inthe CSF of patients suffering from herpes simplex 1 en-cephalitis. Also, measles virus has been shown to induce MCP-1 and MCP-2 in human monocytes. (35) Recently, we showedthat an influenza A virus infection of human monocytes inducedMCP-1 and IP-10.(36) Another viral induction of IP-10 was ob-served in mouse astrocytes and microglia after infection withNewcastle disease virus.(37) Several recent reports describe IP-10 as an LPS-inducible protein, for example, in U937cells,(3 8,39) in the murine macrophage cell line RAW264.7, (40,41) and in human monocytes. (42 ) We were unable toconfirm the last results, as our study clearly shows that LPS ex-erts inhibitory effects on IP-10 expression in human monocytes.

As a first approach to explore the underlying molecularmechanisms, we examined chemokine gene expression after in-hibition of de novo protein synthesis with CHX. The tran-scriptional mechanism s of MCP-1 and IP-10 expression werefound to be completely different. MCP-1 expression of LPS-stimulated or VSV-exposed human monocytes was entirelyabolished by CHX treatment, whereas the opposite result wasobtained for IP-10 (Fig. 6). On the one hand, the expression ofMCP-1 appeared to be obligatorily dependent on de novo pro-tein synthesis, and, on the other hand, IP-10 expression was en-tirely independent. A posttranscriptiona l regulation of mRNAstability may be additionally possible. Thus, IP-10 is apparentlyinducible without prior induction of IFN-g , in the typical man-ner of an immediate-earl y gene.

In conclusion, our results show that the inducible expressionof IP-10 and MCP-1 escapes the general inhibition of proteinsynthesis by VSV. This supports our suggestion that most vi-

ral infections of human monocytes exclusively induce MNC-attracting chemokines, (12) which offers an explanation for thewell-known fact that virus-infected tissue is characterized byan MNC-dominated leukocyte infiltration.

ACKNOWLEDGMENTS

This work was supported by a grant from the DeutscheForschungsgem einschaft (Sp 395/2-2). We thank the Depart-ment of Transfusion Medicine, University of Marburg, for do-nating the buffy coats.

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Address reprint requests to:Dr. Diethard Gemsa

Institute of ImmunologyPhilipps University

Robert-Koch-S tr. 17D-35037 Marburg

Germany

Tel: 01149-6241-2866 492Fax: 01149-6421-2866 813

E-mail: gemsa@ mailer.uni-mar burg.de

Received 4 October 1999/Accepted 13 March 2000

VSV-INDUCED CHEMOKINES 621