Journal of Neuroimmunology, 37 (1992) 169-176 169 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00

JNI 02153

Monocytes in active multiple sclerosis: intact regulation of HLA-DR density in vitro despite decreased HLA-DR density

in vivo *

Richard M. Ransohoff ~.b, Vincent K. Tuohy b,c, Barbara P. Barna d and Richard A. Rudick b,e

Department of Molecular Biology, b Department of Neurology (Mellen Center For Multiple Sclerosis Treatment and Research), ' Department of General Medical Sciences, a Department of Immunopathology, Cleceland Clinic Foundation, Cleceland, OIL USA

(Received 29 July 1991) (Revised, received 29 October 1991)

(Accepted 4 November 1991)

Key words: Multiple sclerosis; Class II major histocompatibility complex; Monocytc; Interferon

Summary

HLA-DR expression on circulating monocytes varies as a function of disease activity in patients with multiple sclerosis (MS), a putative immunopathological demyelinating disorder. Specifically, monocytes isolated from subjects with active MS exhibit reduced HLA-DR antigen density, and immunoregulatory aberrations such as impaired T lymphocyte-mediated suppression correlate strongly with this quantitative defect. To address the mechanism underlying this phenomenon, we compared in vitro regulation of HLA-DR by interferon beta (IFN/3), interferon gamma (IFNy), and lipopolysaccharidc (LPS) in monocytes from patients with stable and active MS and normal individuals, lnterferon-y and LPS enhanced monocyte expression of HLA-DR equally in both MS patient groups, suggesting that underex- pression of HLA-DR in active MS was not explained by impaired in vivo monocyte responsiveness. Furthermore, interferon regulation of HLA-DR in normals and stable MS subjects was indistinguishable, indicating that aberrant interferon-mediated regulation of class II major histocompatibility complex (MHC) on circulating monocytes does not appear to be a characteristic of the MS disease state.

Introduction

Multiple sclerosis (MS) is characterized by multifocal inflammatory demyelination and sev-

Correspondence to: R.M. Ransohoff, The Research Institute, Department of Molecular Biology, Cleveland Clinic Founda- tion, Cleveland, OH 44195, USA * Presented in part at the l l4tb Annual Meeting of the

American Neurological Association, New Orleans, LA, USA. 1989.

eral lines of evidence have suggested that im- munopathological tissue injury may contribute to lesion formation (Ransohoff, 1991). Genetic sus- ceptibility to MS has been associated with partic- ular class 1I major histocompatibility complex (MHC) haplotypes (Hauser et al., 1989; Vartdal, 1989), compatible with the possibility that the primary structure of the class II MHC antigens (and thus their affinity for specific antigenic pep- tides) may in part determine the occurrence of autoimmune demyelination. Individual class I1 MHC antigens are composed of a/[3 hetero-

170

dimcrs. In the human HLA system, class 11 MHC antigens are termed HLA-DR, H1,A-DQ and HI,A-DP. Class 1I MHC antigen expression is intricately regulated by developmental (Benoist and Mathis, 1990), tissue-specific (Barna et al., 1989), hormonal (Snyder ct al., 1982; Lu et al., 1984; Tripp et al., 1986) and genetic (Massa ct al.. 1987) influences.

Several observations suggest that quantitative aspects of class I1 MHC expression by monocy tc / macrophage lineage cells could bc a factor in the pathogcnesis of MS: (1) H L A - D R antigen expres- sion is significantly rcduccd on monocytes from patients with active compared with stable MS (Baxcvanis ct al., 1989), and immunoregulatory aberrations characteristic of MS correlate with this undcrcxprcssion (Baxevanis ct al., 1990); (2) IFNy administration in an open clinical trial in- duced cxaccrbations of MS and increased the number of circulating H L A - I ) R + / M 3 + cells (Pa- nitch ct al., 1987a,b); (3) in chronic-active MS lesions, a high degree of class I1 MHC expression is present on microglial cells (Woodroofc c t a l . , 1986). In these reports, the relation of monocyte H L A - D R to disease activity appears to be incon- sistent, since active disease is associated with increased H L A - D R in paticnts receiving IFNy, but correlates with decreased H L A - D R in a study of untreated subjects. This discrepancy may bc accounted for by additional immune stimulation by IFNy in patients receiving this agent.

Studies performed to date have left unresolvcd whether circulating monocytcs in MS patients with active disease undercxprcss H L A - D R anti- gcns because of impaired responsiveness to intcr- ferons, which arc the primary regulators of class II MHC on monocy tc /mac rophage lineage cells. Therefore, we evaluated baseline and interferon (IFN)-induccd HLA -DR expression on highly pu- rified peripheral blood monocytes isolated from individuals with either clinically active or clini- cally stable MS and a control population. We observed significant differences in spontaneous monocyte H L A - D R expression between patients with active MS and either normal individuals or stable MS patients, consistent with prior observa- tions and compatible with the concept that mono- cytc HLA-DR expression varies as a function of disease activity. These results were not likely at-

tributablc to impaired monocyte responses to en- dogenous stimuli since in vitro regulation of HI~A-I)R expression by IFNy and LPS were in- distinguishable among the various groups.

Materials and methods

Study participants Patients were selected from the Mellon Center

for Muhiplc Sclerosis Treatment and Research based on clinical fcaturcs and their willingness to donate a sample of blood. All patients met re- search criteria for definite MS (Poser et al., 1983). Peripheral blood monocytes wcrc isolated from patients who were stable (stable MS, n = 9) or experiencing acute or subacutc exacerbations (ac- tive MS, n = 9). Healthy controls (normals, n = 9) matched for age were studied as a comparative group. None of the MS patients or healthy blood donors had ingested corticosteroids, salicylatcs, or non-steroidal anti-intlammatory drugs. MS pa- tients were categorized as active or stable prior to monocyte studies. The characteristics of these 27 patients have bccn reported previously (Rudick and Ransohoff, 1991). Briefly, all patients in thc active MS group had new symptoms accompanied by objective worsening of pre-existing neurologi- cal impairments or by new signs on neurological examination. The patient groups and control group were well matched for age and gendcr, and the MS groups were matched for disease duration and Kurtzke Expanded Disability Status Score (Kurtzkc, 1983). The mcdian duration of new symptoms in the active MS group was 4 wceks.

Isolation and culture of monocytes Monocytes were isolated from EDTA-anti-

coagulated whole blood. Blood was brought to a final concentration of 10% dextran. After red blood cells had sedimentcd, white blood cells remaining in the plasma were separated by een- trifugation at room temperature on Nycodenz medium (Accurate Chemical, Westbury, NY). Cells layering at the interface were transferred by pipette to centrifuge tubes, washed twice in Hanks ' balanced salt solution without Mg 2~ or Ca 2" and counted. The concentration was ad- justed to 3 x 10S/ml after which cells were trans-

ferred to plastic tissue culture flasks in a total volume of 5 ml RPMI-1640 (Gibco, Grand Island, NY) medium supplemented with 10% human AB serum (Flow Labs-ICN, Costa Mcsa, CA) and 1% penicil l in/ s t reptomycin/ t.-glutamine. After al- lowing cells to adherc for 60 min at 37°C, non-ad- herent cells werc removed by washing the flasks four times with RPMI-1640 at 37°C. This procc- dure resulted in a highly purificd prcparation of adherent monocytes, since > 95% of cells wcrc positive for non-specific csterase by cytochemical stain. Lcss than 3% of ceils were positive by FACS analysis using monoclonal antibodies reac- tivc with CD2 (T cclls) or CD19 (B cells). Media wcrc free of cndotoxin by Limulus Amoebocyte Lysatc assay (Woods Hole, MA) (sensitivity < 0.3 ng/ml).

Intetferons Immunoaffinity-purificd natural IFNy (3 × 108

I U / m g protcin) was obtained from Green Cross (l_x)s Angeles, CA). Recombinant IFN/3 (2 × 108 I U / m g protein) was provided by Biofcron Bio- chcmische Substanzen GmbH & Co, Lauphcim, F.R.G. This mammalian cell line product is glyco- sylated and has an amino acid sequence identical with naturally occurring IFN/3 (Reiscr and Hauscr, 1987). Biological activity of interferon prcparations was confirmed by encephalomy- ocarditis virus yield reduction assays (Jameson et al., 1977).

Monoclonal antibodies Anti-Leu-5b (CD2) conjugated to fluorescein

isothiocyanate (FITC) (Becton-Dickinson) was used as a pan T-cell marker; anti-Lcu-12 (CDI9) conjugated to FITC (Becton-Dickinson) was used as a pan B-cell marker; and ant i-HLA-DR conju- gated to phycoerythrin (PE) (Becton-Dickinson) was used to determine HLA-DR antigen expres- sion. Irrelevant murine antibodies of the same isotypes (IgG1, lgG2a) conjugated to FITC and PE, respectively, were used in all experiments. Monoclonal antibody Leu-M3, a human mono- cyte marker that reacts with CD14, was not used because cultured monocytes showed decreased expression of CD14 with time in culture (data not shown). Since cells werc greater than 95% posi- tive for non-specific esterase, cells negative for

171

CD2 and CD19 but positive for HLA-DR wcre considcred to be monocytes.

Flow cytometry and data analysis Cells wcrc analysed using a fluorescence-

activated cell sorter (FACScan, Bccton Dickin- son, San Jose, CA) to determinc surface pheno- type and HLA-DR antigen density. Our principal interest was in determining HLA-DR antigen density on monocytes. For purposes of data anal- ysis, wc converted peak channcl relative fluorcs- ccnt intensity (RFI) for HLA-DR aquired in a four-dccadc log mode to a linear scale, which simplifies quantitative comparison of fluores- cence intensities for different samples (Muirhead et al., 1983). Group and dose comparisons wcrc made using KruskaI-Wallis, Wilcoxon rank sum, and Wilcoxon signed rank tests and displayed as group median values since the data were not normally distributed. / '-Values are two-sided and are not adjusted for multiplc comparisons.

Results

Spontaneous monocyte HLA-DR expression Monocytes from healthy individuals and pa-

tients with rheumatoid arthritis were reported to exhibit increased HLA-DR expression as a func- tion of duration of tissue culture (Becker, 1984; Hassan et al., 1989). In our prcliminary studics in MS patients and controls, no significant differ- cnces in HLA-DR antigen density were observed in freshly isolated monocytcs compared with monocytes held in culture for 48 h (data not shown). This observation confirmed that the iso- lation protocol efficiently excluded contaminating T lymphocytes, which could serve as sourcc of activating cytokines; furthermorc culture condi- tions did not introduce LPS or othcr stimuli for HLA-DR induction. Thereforc, in subsequent cx- pcriments, spontaneous HLA-DR expression was monitored after 48 h of culture to permit direct comparison with stimulus-induced class II expres- sion.

Median HLA-DR density on monocytes from patients with MS and controls is shown in Fig. 1. HLA-DR density was significantly lower on monocytes isolated from patients with active MS

172

Controls n = 9

Active ~ n=9

Stable ~ n=9

0 100 200 300 400 500

M e . a n Peak ct~nnel RFI

Fig. I. Spontaneous H L A - D R expression by MS and control monocytes. Monocytes were isolated by density gradients fol- lowed by plastic adhcrance and cultured for 48 h. Surfacc l i t ,A-DR density was measured by FACS and expressed as peak channel relative fluorescence intensity. Monocytc HLA- DR density was lower in patients with active MS patients compared with stable MS patients or normal controls (Kruskal-Wallis test P = 0.01; significantly different pairs:

contrt)l, active MS; stahle MS, active MS).

compared with cells from patients with stable MS or normal controls. Stable MS patients did not diffcr significantly from normals.

Induction o[ ItLA-DR expression by IFNT I F N T is thc b e s t - c h a r a c t e r i z e d i n d u c e r of class

II MHC expression and has been demonstrated to upregulate HLA-DR on human monocytes in vivo (Panitch et al., 1987b) and in vitro (Sztcin et al., 1984; Rhodes et al., 1986). Figure 2 shows a rcprescntativc experiment, in which purified monocytes were incubated for 48 h in culture medium with and without IFNT, 10 U / m l . The presence of IFNT was associated with clearly increased HLA-DR density. Exposure to increas- ing doses of natural IFNT augmented HLA-DR density on monocytes from stable MS patients and normal individuals in a dose-dependcnt fash- ion (Fig. 3). HLA-DR density was significantly higher in cultures exposed to natural 1FNT com- pared with medium controls. Subsequent experi- ments using comparable conccntrations of rccom- binant IFNT indicated that this response was induced by the IFNy moiety in the natural IFNT preparation (data not shown).

In experiments to determine whether cells from active MS patients were normally responsive, IFNT was used at a subsaturating concentration of 10 U / m l . At that dose, increased HLA-DR

too

(1) Time 012

(2', T~me 007

(3 )T imeOC8

o . . . . ,2qq , , ,4oq, , 6£,0 , , 8 o q , 79oo

t HLA-DR - - I so tyoe ]FN~T

~, HLA -DR I ,:.~ medium J~j.

10 0 101 10 2 10 3 10 z"

FL2 Fig. 2. Monocyte HLA-DR density in presence or absence ol IFN'),. Monocytcs were isolated from a normal donor and incubated in medium alone or in the presence of IFNT, I(I t J /ml . Peak channel RFI aquircd in a 4-decade log mode was converted to a linear scale as described (Muirhead ct al.. 1983). Linear peak channel RFI was 6 for isotype control, 257 tbr t I I ,A-DR without IFNy ( I ILA-DR medium), and 1843 l,.)r t l I ,A-DR with IFNT. Treatment with IFNT did not change

the isotype peak channel.

antigen density was observed on monocytes from all three groups (Fig. 4) and there were no signifi- cant differences among the three groups in amount of change or final value of IFN"/-induced HLA-DR antigen expression. To support the

Co~lrols I ~ Stable MS rt = 6 n = 7

NO I~N

ILI/rnl

10 IU/ml

tO0 IU/IT~

i i i I

0 ~ 1600 2400 3200 4 0 0 0

Fig. 3. Dose-dependent up-regulation of monocyte I ILA- I )R antigen expression by IFNy. Monoeytes from normal individu- als (solid bars) or stable MS patients (cross-hatched bars) were cultured for 48 h in medium supplemented with IFNT heft)re harvesting and FACS analysis. Exposure to IFNT resulted in significant enhancement of HI ,A-DR antigen den- sity in both groups combined (Wilcoxon signed ranks test P < (I.(101). There were no significant differences between MS patients and controls at any dose level (Wilcoxon rank s u m

test P values: 1 I U / m l , P = 0 .42:10 I U / m l P = (I.42:l(10 I U / m l P = 0.61).

conclusion that pathways for class II MHC induc- tion were intact in monocytes from subjects with active MS, we evaluated HLA-DR induction by LPS, a monocytc stimulus that activates genc expression by mechanisms distinct from IFNs (Hamilton, 1989). Significant increases in HLA- DR antigen dcnsity werc observed in monocytes from all three groups (Fig. 4) and there were no significant differences between patient groups or between MS patients and normal individuals in regard to HLA-DR antigen final value or amount of change from medium control.

I

Induction of HLA-DR expression by 1FN[3 There is evidence that IFN/3 can exert tissue-

spccific effects on class II MHC exprcssion. In particular, exposure to IFN/3 has been reported to inhibit IFN-/-induced expression of class II MHC genes by human astrocytes, astrocytoma cell lines (Barna et al., 1989; Ransohoff et al., 1991), thioglycollate-clicited murine peritoneal macrophages, and macrophage-like cell lines (Ling ct al., 1985; Inaba et al., 1986). In contrast, sevcral laboratories including ours have described up-regulation of HLA-DR density on human monocytes in culturc by high concentrations of

Controls ~ Actlve ~ ~ Stable MS n = 9 n = 9 n ~ 9

IIZN gamr r~

L P S

I r

0 4 8 0 '¢)60 1 4 4 0 1 9 2 0 2 4 0 0

k 4 ~ a n P e a k oh l l r l l ~ l I ~1

Fig. 4. IFN3, and LPS-induced H L A - D R expression in MS patients and controls. Mon~ 'y tes from normal individuals (solid bars), active MS patients (cross-hatched bars), or stable MS patients (hatched bars) were cultured in medium with or without IFNy (10 U / m l ) or LPS (0.1 g g / m l ) for 48 h before harvesting and FACS analysis. Stimulation by both IFN7 and LPS produced significant enhancement of HL A-DR antigen expression compared to medium controls (Wilcoxon signed ranks test P < 0.001). There were no significant differences between patient groups (Kruskal-Wallis test IFNy P = 0.76;

LPS P = 0.26).

~ m l C o n t r o l s ~ S t a b l e I v ~ n = 6 n = 7

173

NO IFN B e t a

1 I1.J/ml

10 tU/ml

1 0 0 IU/ml

i i I I

0 4 0 0 8 0 0 1 2 0 0 1 6 0 0 2 0 0 0

M e . a n P e a k ohannet r ~ l

Fig. 5. Dose-dependent up-regulation of monocyte HLA-DR antigen expression by IFN/3. Monocytcs from normal individ- uals (solid bars) or stable MS patients (cross-hatched bars) were cultured for 48 h in medium supplemented with IFN/3 before harvesting and FACS analysis. Exposure to IFN/3 at 100 I U / m l resulted in significantly higher HLA-DR density in both groups combined (Wilcoxon signed ranks test P < 0.001), and therc werc no significant differences in these stable MS patients compared with normals at any dose level (Wilxcoxon rank sum test P-values: 1 I U / m l P = 0.68:10 I U / m l P = 0.94:

100 I U / m l P = (I.35).

recombinant IFNa or IFN/3 (Sztein ct al., 1984: Ransohoff et al., 1991), and analogous in vivo effects of IFN/3 have been reported (Spear et al., 1987).

We incubated monocytes from controls and stable MS patients with varying concentrations of IFN/3 to evaluate effects of MS disease-state on class 1I MHC regulation by IFN/3 (Fig. 5). Mono- cyte HLA-DR antigen density was not signifi- cantly affected by exposure to IFN/3 at 1 or 10 U / m l in either patients or controls. However, at a dose of 100 U/ml , there was a significant increase in HLA-DR antigen density. Subsequent experiments using higher concentrations of IFN/3 confirmed significantly increascd HLA-DR anti- gen density by IFN/3-treated monocytes (data not shown).

Discussion

Experiments described in this report were per- formed to address the relationship between the regulation of monocyte class II MHC and the MS disease-state. In particular, we analysed mono- cyte HLA-DR expression for several reasons: (1)

174

monocyte HLA-DR expression has been reported to be diminished in patients with active MS (Baxcvanis c t a l . . 1989); (2) reduced monocyte HLA-DR density has been strongly correlated with immunoregulatory aberrations such as im- paired autologous mixed lymphocyte reaction and antigcn-nonspecific suppression (Baxevanis et al., 1990); (3) monocyte HLA-DR expression can be modulated in vitro and in vivo by IFNs, which are of potential therapeutic and pathophysiologica[ interest in MS.

In this study, cell-culture experiments were used to determine if decreased monocyte HLA- DR expression in vivo could be correlated with an impaired response to IFN induction of class I1 MHC in vitro. Monocyte culture conditions used in these experiments did not enhancc class II MHC density so that spontaneous and IFN-in- duced HLA-DR expression could be directly comparcd. We observed that spontaneous HLA- DR antigen expression by monocytes was de- pressed in active MS compared with eithcr stable MS or normal individuals. These data are consis- tent with rcsults reported by Baxevanis (Baxevanis et al., 1989). Diminished HLA-DR by monocytes from active MS subjects could not be explained by an intrinsic MS-related class II MHC regula- tory defect, since patients with stable disease who were otherwise indistinguishable from active pa- tients had expressed levcls of monocyte HI.A-DR comparable to those observed in normal individu- als.

It should be noted that median duration of symptoms in patients classed as clinically active in the present report was 4 weeks. It may be pro- posed that the characteristics observed in our study of monocyte MHC class II expression are representative of the recovery period after MS cxacerbation, rather than the acute events in- volved with new symptom occurrence. Our paral- lel investigation of monocyte PGE 2 secretion in this patient population provides some support for this viewpoint (Rudick and Ransohoff, 1991). Longitudinal investigations will be rcquired to define sequential alterations in monocyte HI.A- DR expression during the evolution of an MS exacerbation.

Decreased HLA-DR density on monocytes of active MS patients could not be accounted for by

a diminished capacity to express HI.A-DR, since IFNy treatment induced HI.A-DR antigen ex- pression to an equal extent in cells from all groups tested. LPS exerted similar effects on cells from all three groups, inducing modest enhance- ment of HI.A-DR expression. Therefore, reduced monocyte HLA-DR antigen density seen in active MS is not likely attributable to altered monocyte responsivity. It seems more likely that this obser- vation is due to reduced IFN production by MS leukocytes (Neighbor, 1984) or inhibitory influ- ences of other immunoregulatory cytokines. Po- tentially, increased concentrations of leukocyte- derived immunoregulatory factors such as prosta- glandins or TN F a could account for decreased monocyte HEA-DR expression in subjects with active MS (Watanabe and Jacob, 1991; Snyder el al., 1982). In this regard, we evaluated monocyte production of TN F a and PGE 2 in a companion study to the present report (Rudick and Ranso- hoff, 1991). In patients with active MS, "sponta- neous' (unstimulated) secretion of both monocyte products was reduced. Therefore, there is no direct evidence to suggest a relationship between mono(o, te synthesis of these components and re- duced monocyte HLA-DR antigen density in ac- tive MS. Furthermore, a recent longitudinal study demonstrated that serum TN F a levels were in- distinguishable in active and stable MS patients (Sharief and Hcntges, 1991). Accordingly, it re- mains unclear what regulatory factors lead to decreased HLA-DR expression in monocytes from patients with active MS.

The ability of IFN/3 to enhance monocytc class 11 MHC expression was noteworthy, since IFN~ has been reported to strongly inhibit class II MHC induction in several other cell types includ- ing murine thioglycollate-elicited peritoneal mac- rophages (Ling et al., 1985; Inaba et al., 1986). These divergent responses to IFN/3 probably re- sulted from different states of differentiation or activation of the monocyte /macrophage cells be- ing studied. Support for the notion of differentia- t ion-dependent regulation of monocyte /macro- phage MHC class I1 expression by cytokines comes from a recent study by Watanabe and Jacob (1991). In this study, contrasting reports of the effects of TN F a on IFNy-induced monocyte MHC class II expression could be rationalized by

the demonstration of maturation-dependent ef- fccts of the cytokine: TNFo~ was shown to stimu- late MHC class II induction in immaturc HL-60 monoeytic cells, but to inhibit HLA-DR expres- sion in ceils which had becn induced to diffcren- tiate with TPA. We speculate that similar differ- entiation-dependent responses to IFN/3 could ac- count for the disparate effects on monocytc/ macrophage MHC class I1 induction which have been rcported. In this regard, IFN/3 has bccn dcscribed as an inhibitor of MHC class II expres- sion only in studies of activated macrophagcs, while induction of MHC class II has bcen rc- ported in circulating monocytes or cells freshly cultured from the vascular compartment.

Despitc the similarity of IFN-,/ and IFN/3 in stimulating HLA-DR density on monocytes in vivo (Spear et al., 1987; Panitch et al., 1987b), MS trcatment trials produced strikingly disparate clinical rcsponscs to IFNy and IFN/3, with the former producing dctrimental (Panitch et al., 1987a) and thc latter bencficial (Jacobs et al., 1986) results. IFN/3 has bccn reported to aug- mcnt mitogen-driven T ccil suppression in vitro (Noronha et al., 1990); it is plausiblc that the beneficial cffect of IFN¢3 could be mediated in part through enhancement of monocyte HLA- DR, given the experimental results described by Baxevanis and colleagucs. It is possible to specu- late that IFN/3 might generate two types of bene- ficial alterations in HLA-DR expression in MS: enhanced monocyte HLA-DR with concomitant incrcased T cell-mediated suppression, and de- creascd HLA-DR expression on astrocytes and possibly macrophages at tissue sites of inflamma- tion.

Acknowledgements

This work was supported by National Multiple Sclerosis Society RG-2019-A-I (RAR). Dr. Ran- sohoff and Dr. Tuohy are Harry Weaver Scholars of thc National Multiple Sclerosis Society and arc supported by NS-29095-01 (VKT)and K08-01265 (RMR) from the NINDS. We gratefully acknowl- edge the support of the Multiple Sclerosis Women's Committee. We thank Shari Meden-

175

dorp for statistical consultation, Ms. Barbara Ja- cobs and Dr. Gabriel Stancscu for technical assis- tance, and Amy Guild for secretarial support.

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