Fish & Shellfish Immunology (1998) 8, 545–553Article No. fi980158

Modulation of MHC class II expression in rainbow troutOncorhynchus mykiss macrophages by TNFá and LPS

JOHN KNIGHT1†, RENE J. M STET

2AND CHRISTOPHER J. SECOMBES

1*

1Department of Zoology, University of Aberdeen, Aberdeen AB24 2TZ, U.K.2Cell Biology and Immunology Group, Wageningen Institute of Animal

Sciences, Wageningen Agricultural University, The Netherlands

(Received 18 March 1998, accepted 20 May 1998)

Modulation of MHC class II â chain expression on head kidney macrophageshas been demonstrated. Following in vitro stimulation for 24 h, 48 h or 72 hwith human recombinant TNFá plus LPS increased numbers of MHC class IIpositive macrophages were found relative to control cells, by FACS analysiswith an anti-trout MHC class II â chain serum. Northern blot analysisconfirmed that increased transcription was occurring. That TNFá synergisedwith LPS was also clearly demonstrated using Northern blots, where TNFáinduced no e#ect whilst LPS alone had a small stimulatory e#ect. A timecourse experiment demonstrated that 4 h post-stimulation was an optimal timeto isolate macrophage RNA post stimulation with TNFá and LPS, with latertimes showing less clear modulation of MHC class II â expression. Lastly, adose response experiment performed with LPS alone revealed that only small(if any) e#ects were apparent when using 6·25 and 12·5 ìg ml"1, whereas 25and 50 ìg ml"1 were equally stimulatory. The mechanisms and consequencesof modulating MHC class II expression are discussed. ? 1998 Academic Press

Key words: MHC class II â chain, rainbow trout, macrophages, tumornecrosis factor á, LPS.

†Present address: School of Biological Sciences, University College of Swansea, SingletonPark, Swansea, U.K.

*Author to whom correspondence should be addressed.

I. Introduction

The essential components necessary for an adaptive immune system, MHCmolecules, TCR and Ig, appear to have arisen in the earliest jawed vertebrates(Bartl et al., 1997; Rast et al., 1997; Schluter et al., 1997). The geneticmechanisms that generate diversity of Ig and TCR in fish have been wellstudied in recent years (Litman, 1996; Rast et al., 1997), and analysis of thepolymorphism of fish MHC molecules has begun (Miller & Withler, 1996;Okamura et al., 1997). However, factors that influence the expression of thesemolecules and the mechanisms by which they are regulated are largelyunstudied in fish.

With respect to MHC molecules, class I and class II genes are present inboth cartilaginous and bony fish (Stet et al., 1996; Bartl et al., 1997), as is thegene for â2-microglobulin (Dixon et al., 1993). Since populations of leucocytes

5451050–4648/98/070545+09 $30.00/0 ? 1998 Academic Press

546 J. KNIGHT ET AL.

with specific helper and cytotoxic activity are known in fish (Clem et al., 1991;Hasegawa et al., 1998), and potential CD4/CD8 binding domains have beenidentified in the MHC sequences (Stet et al., 1996; Rode, 1997), it is likely thatMHC restricted T cell subpopulations will be present. In mammals, a criticalstep in both specific humoral and cell-mediated immune responses is therelease of cytokines from helper T cells, which itself is dependent uponappropriate presentation of peptides by MHC class II molecules on macro-phages, dendritic cells and B cells (Rohn et al., 1996). Whilst much has to belearnt about antigen presentation in fish, it is known that macrophages and Bcells express MHC class II transcripts (Rodrigues et al., 1995) and that theycan function as antigen-presenting cells in a seemingly alloantigen-restrictedfashion (Vallejo et al., 1992).

Since expression of MHC class II genes is a key factor in T cell stimulation,their expression is tightly regulated (Mach et al., 1996). Whilst constitutiveexpression on B cells and macrophages occurs, many soluble factors canmodulate MHC class II expression in these cells, or induce expression in MHCclass II negative cells (Glimcher & Kara, 1992). The present study addresseswhether MHC class II expression can be modulated in fish macrophages, usinga recently developed anti-trout MHC class II â chain serum (van Lierop et al.,1998) and Northern blot analysis. Both TNFá and LPS have been used tostimulate the macrophages, since it has been shown previously that suchstimuli are particularly potent signals to up-regulate trout macrophagefunction (Campos-Perez et al., 1997). No attempt was made to distinguishbetween possible di#erent MHC class II genes in trout since in most fishspecies studied to date, including trout, the data suggest that only onefunctional DAB locus is present (Glamann, 1995; Godwin et al., 1997).

II. Materials and Methods

FISH

Rainbow trout Oncorhynchus mykiss, 300–500 g, obtained from a local fishfarm were maintained in aerated fibreglass tanks supplied with a continuousflow of recirculating, dechlorinated water, at 14–16) C. Fish were fed twicedaily on commercial trout food (EWOS).

MODULATION OF MACROPHAGE MHC CLASS II PROTEIN EXPRESSION

Head kidney macrophages were isolated from trout using standard proto-cols (Secombes, 1990). In brief, head kidney cell suspensions were fractionatedon 34%/51% Percoll density gradients and the interface cells collected.Macrophage-enriched suspensions from individual trout were split into two orfour aliquots and plated out in 10 cm tissue culture plates (Nunc) to give afinal density of 1#107 macrophages per dish. After 3 h in culture non-adherentcells were removed and the macrophages were cultured overnight prior toin vitro stimulation with human recombinant tumor necrosis factor á (TNFá,12·5 iu ml"1, R & D Systems) and E. coli lipopolysaccharide (LPS, 25 ìg ml"1,Sigma), determined to be optimal for macrophage stimulation in previous

MODULATION OF MHC 547

studies (Campos-Perez et al., 1997). Control dishes contained media only. Alldishes were then incubated at 18) C for 24 h, 48 h or 72 h. Cells were subse-quently harvested by scraping the dishes and stained for FACS analysis usingthe anti-rainbow trout class II â polyclonal antiserum (van Lierop et al., 1998).The cells were washed twice and resuspended in phosphate bu#er (PBS)containing bovine serum albumin (BSA, 1%) and sodium azide (0·1%). Theanti-class II serum was then added to give a final dilution of 1:1200, and thecells placed on ice for 30 min. The cells were then washed (twice), resuspendedin PBS/BSA/azide and fluorescein isothiocyanate labelled donkey anti-rabbitIg serum (SAPU, Carluke) added to give a final dilution of 1:50. After 30 minon ice the cells were washed as above, resuspended in 1 ml PBS/azide and 5 000cells per sample analysed with an EPICS-C flow cytometer (Coulter). Thecursor was set to 1% positive cells using control cells incubated without theprimary antibody. Up- or down-regulation of MHC class II expression wasmeasured relative to the control cultures, and tested statistically withStudent’s t-test. Cells from five fish were examined for each parameterinvestigated.

MODULATION OF MACROPHAGE MHC CLASS II mRNA EXPRESSION

To determine whether modulation of MHC class II expression was undertranscriptional control, and to confirm the data obtained with the polyclonalanti-trout MHC class II serum used above, Northern blot analysis of MHCclass II mRNA levels was studied. As for the FACS analysis, macrophageswere isolated and cultured overnight prior to stimulation in vitro with TNFáand LPS. In initial experiments, cells were incubated with 25 iu ml"1 TNFáand/or 25 ìg ml"1 LPS for 4 h, after which total RNA was extracted usingRNAzol (Laing et al., 1996). Following on from these experiments, a timecourse experiment was performed to confirm when the increase in expressionwas maximal, stimulating the macrophages for 1, 2, 4 or 8 h. Lastly, a doseresponse experiment was performed with LPS using 6·25, 12·5, 25 and 50 ìgml"1 for 4 h. Cells from a minimum of three fish were examined for eachparameter investigated.

For Northern blot analysis, a Dig-labelled 30-mer antisense RNA probe (5*-CTTCTCTCCAGACTTGGGTGTGTACTCCAG-3*) was synthesised (Genosys)based on the published MHC class II sequence for rainbow trout (Glamann,1995). This probe gave a single, strong band of 1·6 kb in Northern blots andnothing with negative controls, and was used for all the experiments describedbelow. Twenty-five ìg of RNA was loaded per lane of a 1% agarose gel.Following electrophoresis at 90 v for 3–4 h in 1#MOPS and vacuum transferto a N+ nylon filter (Amersham), the membrane was baked for 1 h at 80) C andprehybridised for 1 h at 45) C. Hybridisation with the Dig-labelled probe wasthen performed overnight at 42) C, followed by washing once with 2#SSC/0·1% SDS for 20 min and twice with 0·2#SSC/0·1% SDS for 15 min, at 60) C.The Dig-labelled probe was then detected with an alkaline phosphatase-labelled anti-Dig serum (Boehringer and Mannheim), followed by the chemi-luminescence substrate CSPD, as described by the manufacturers. Bands werefinally visualised with X-ray film using an exposure time of approximately 2 h.

548 J. KNIGHT ET AL.

In all cases, a Dig-labelled â-actin probe (Laing, 1996) was used as a controlto ensure that any increases seen were not a result of a general increasein mRNA. In some cases the relative levels of mRNA were quantified bydensitometric scanning of exposed film, using a UVP gel imaging system andUVP Gelworks ID advanced software, and expressed relative to the controlvalues.

50

0

Hours post-stimulation

% M

HC

cla

ss I

I po

siti

ve c

ells

72

40

30

20

10

24 48

Fig. 1. Percentages of MHC class II positive head kidney macrophages, as detected byFACS analysis after reaction with an anti-trout MHC class II â chain serum,following culture for 24 h, 48 h or 72 h in the presence (stimulated, /) or absence(control, .) of TNFá and LPS. Data are presented as means&S.E. for five fish.

III. Results

Between 7–10% of control head kidney macrophages expressed MHC classII molecules, as detected by FACS analysis after reaction with the polyclonalanti-MHC II â chain serum (Fig. 1). After stimulation of macrophages withTNFá and LPS for 24 h, 48 h or 72 h, significantly more (P<0·05) macrophageswere MHC class II positive, as detected by a marked increase in fluorescence(Fig. 2), with highest numbers being detectable after 72 h (Fig. 1). Stimulationof macrophages with TNFá and LPS also increased MHC class II â chainmRNA expression relative to levels in control cells, as detected by Northernblot analysis with samples taken 4 h post-stimulation (Fig. 3). That the TNFásynergised with LPS to increase expression was also clearly demonstratedusing Northern blots (Fig. 4a,b). TNFá had no e#ect on MHC class II mRNAlevels and LPS alone had a small stimulatory e#ect but together there was aclear increase in expression. A time course experiment using RNA frommacrophages stimulated with TNFá and LPS, demonstrated that 4 h post-stimulation gave the highest increase in MHC class II â chain expression, withlater times showing less clear modulation of expression (Fig. 5). Lastly, a doseresponse experiment was performed with LPS, to determine whether otherconcentrations of LPS may be more e#ective for in vitro stimulation. This

MODULATION OF MHC 549

experiment revealed that only small, if any, e#ects were apparent when using6·25 (data not shown) and 12·5 ìg ml"1 LPS whereas 25 and 50 ìg ml"1 wereequally stimulatory (Fig. 6). No significant increase in expression of â-actinmRNA was observed post-stimulation in any of the above experiments, asshown in Fig. 4.

Log fluorescence

Cel

l nu

mbe

r

Control

Log fluorescence

TNF/LPS stimulated

Fig. 2. FACS analysis of rainbow trout head kidney macrophages from a represen-tative fish, after culture for 48 h in the presence or absence of TNFá and LPS, andreaction with an anti-trout MHC class II â chain serum. Note the marked increasein fluorescence in the stimulated cells relative to control cells.

C S C S C S

Fig. 3. Northern blot analysis of MHC class II â chain expression after stimulation oftrout head kidney macrophages from three di#erent fish with (S) or without (C)TNFá and LPS for 4 h.

IV. Discussion

This study has confirmed that MHC class II â chain expression in rainbowtrout macrophages can be modulated by cytokines and pathogen-derived(LPS), with potential functional consequences for antigen presentation andstimulation of specific immunity. Both mRNA and protein levels wereincreased post-stimulation with TNFá and LPS, the latter detectable as anincrease in the number of cells positive by FACS analysis. TNFá alone wasunable to increase expression in macrophages but was able to synergise withLPS. This is similar to previous studies of other fish leucocyte functions whereTNFá has little e#ect unless added with a second signal (Hardie et al., 1994;Jang et al., 1995a). This may relate to the fact that human TNFá was beingused since fish TNF has still to be cloned, although biological activities akinto TNF are known (Jang et al., 1995b). However, in mammals TNFá alone alsohas little e#ect on MHC class II levels but synergises with IFN-ã to enhanceexpression (Chang & Lee, 1986; Panek et al., 1992, 1994). In contrast LPS was

550 J. KNIGHT ET AL.

1.8

0.8

Exp

ress

ion

rel

ativ

e to

un

stim

ula

ted

con

trol

LPS

1.6

1.4

1.2

1.0

Control TNF TNF/LPS

MHC class II

β-actin

TNF/LPS

LPSTNF

Contro

l

(b)

(a)

Fig. 4. Northern blot analysis of MHC class II â chain expression (/) and â-actinexpression (.), after stimulation of trout head kidney macrophages with TNFá, LPSor both TNFá and LPS for 4 h. Control=unstimulated macrophages. (a) Exposedfilm; (b) densitometric readings of the film in (a) expressed relative to MHC class IIor â-actin control lanes.

C 1 h C 2 h C 4 h C 8 h

Fig. 5. Northern blot analysis of MHC class II â chain expression after stimulationof trout head kidney macrophages with TNFá and LPS for 1, 2, 4 or 8 h.C=unstimulated control macrophages. Note that each pair of stimulated andcontrol lanes is from a separate fish. The largest increase in expression is seen 4 hpost-stimulation.

an e#ective stimulus alone, although the increase in expression was relativelysmall. Precedents for LPS increasing and decreasing MHC class II expressionare known in mammals (Glimcher & Kara, 1992), dependent upon cell type andposition in the cell cycle.

The increase in mRNA expression seen is most likely a consequence ofthe increase in the number of macrophages transcribing the MHC class II â

MODULATION OF MHC 551

gene(s), since MHC class II expression is primarily at the level of transcrip-tion (Mach et al., 1996). Nevertheless, it is also possible that up-regulation ofgene transcription in macrophages already expressing MHC class II couldhave contributed to the increase seen. In mammals TNFá induces a DNA-protein complex, the TNFá-induced complex (TIC-X), that has specificity forthe X-element in the class II promoter (Panek et al., 1992), promotingtranscriptional activation. Since three cis-acting elements are required forMHC class II expression (W, X and Y), other factors may interact with TIC-Xto bring about gene expression, explaining the synergy seen with di#erentsignals.

Previous studies looking at di#erential expression of MHC class II genes infish have shown varying levels of transcription in di#erent tissues and cells(Rodrigues et al., 1995; Koppang et al., 1998). Studies of the ontogeny of MHCclass II expression in carp Cyprinus carpio, using RNA extracted from wholeembryos, have shown that transcription increases with time (Rodrigues et al.,in press). Furthermore, increased MHC class II expression has been notedin the spleen and gills of Atlantic salmon Salmo salar following vaccinationwith Apojet 3-fural (containing Aeromonas salmonicida, Vibrio anguillarumand Vibrio salmonicida), with induction of expression occurring in the heart,liver and head kidney (Koppang et al., 1998). Together with the present study,such findings suggest that MHC class II expression in fish is also tightlyregulated. Whether increased expression correlates with an increased immuneresponsiveness will be important to determine.

This work was supported by a contract from the EC (No. AIR2-CT34-1334). Thanks goto Mr Julian Ashby for help with the FACS analysis, and to Dr Jun Zou for help withanalysis of the Northern blots.

C

12.5

S C

25

S C

50

S

LPS (µg ml–1)

Fig. 6. Northern blot analysis of MHC class II â chain expression after stimulation oftrout head kidney macrophages with varying doses of LPS for 4 h. C=unstimulatedcontrol macrophages, S=LPS stimulated macrophages. Note the clear increase inexpression using 25 and 50 ìg ml"1 but the marginal increase with 12·5 ìg ml"1.

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