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Clin Exp Immunol. Mar 2002; 127(3): 430–435.
PMCID: PMC1906311

The arthritogenic adjuvant squalene does not accumulate in joints, but gives rise to pathogenic cells in both draining and non-draining lymph nodes

Abstract

A single intradermal injection of the adjuvant-oil squalene induces T cell-mediated arthritis in DA rats. The chain of events leading from non-specific provocation of the immune system to arthritis, with clinical similarities to rheumatoid arthritis, is largely undetermined. Here, we combined in vivo tracking of tritium-labelled squalene with lymph node (LN) cell transfer experiments to determine where critical activation events may take place. The majority of squalene remained at the injection site (79%). The amounts recovered in peripheral joints (<1%) were equal to that recovered in other organs that can be targets in autoimmune diseases. This argues that arthritis does not develop as a consequence of adjuvant accumulation in joints. In contrast, substantial amounts of squalene were recovered in hyperplastic LN draining the injection site (1–13%). The adjuvant was deposited to a larger extent in cells than in extracellular matrix. The draining LN cells could transfer arthritis to naïve irradiated DA rats following in vitro stimulation with conA. Interestingly, non-draining LN were also hyperplastic and harboured arthritogenic cells, although they contained low amounts of squalene (<1%). Consequently, the amount of arthritogenic adjuvant in a particular LN is not closely linked to the development of pathogenic cells. The distribution pattern of squalene was similar in MHC-identical but arthritis-resistant PVG.1AV1 and LEW.1AV1 rats, and it was unaffected by T cell depletion with a monoclonal antibody (R73). Thus, T cells and non-MHC genes do not regulate dissemination of squalene, but rather determine arthritis development at the level of adjuvant response.

Keywords: adoptive transfer, arthritis, squalene, tracking

INTRODUCTION

Rheumatoid arthritis (RA) is a chronic, inflammatory joint syndrome that is often suggested to be an autoimmune disease [1,2]. The pathogenesis remains largely unknown, however, which obscures the development of rational cure and prevention [3,4]. Experimental RA-models may yield new knowledge concerning both epidemiology and pathogenesis. One potentially interesting observation is that arthritis can be induced in disease-prone rat strains by a large variety of molecules that non-specifically activates the immune system [5]. This includes synthetic immunostimulating compounds such as avridine [6], microbial cell wall structures such as muramyl dipeptide [7,8], and different types of oils such as pristane from plants [9] and squalene from animals [5,10]. These observations in the rat spurred the recent identification of an epidemiological association between RA and oil exposition (Klareskog and co-workers, unpublished). RA is a clinically heterogeneous disease that is diagnosed when four out of seven inclusion criteria are fulfilled. Direct application of these criteria in rats [10] suggest that adjuvant can induce arthritis that fulfils at least four criteria. Thus, squalene (C30H50), and probably also pristane and avridine [1012], can trigger long-lasting symmetric arthritis involving a minimum of three groups of joints, including hand joints, and that leads to bone erosion. In addition, the clinical manifestations are mediated by T lymphocytes and are controlled by genes both within and outside the MHC [1012]. These similarities to an organ-specific autoimmune disease are enhanced by the fact that inflammation is restricted to peripheral joints [1012]. Several hypotheses may account for this joint restriction, including selective accumulation of adjuvants to peripheral joints. This hypothesis has not been tested, possibly because it requires quantitative determinations that are difficult to achieve for most adjuvants. Here we tested the hypothesis by performing a quantitative kinetic in vivo tracking of tritium-labelled squalene oil. Weighing of lymph nodes (LN) and transfer of LN cells were performed in parallel to give information on squalene-induced changes in the lymphoid system. To determine whether T cells and non-MHC genes influence the parameters mentioned above, experiments were also performed in T cell depleted DA rats and in MHC-identical but arthritis-resistant LEW.1AV1 and PVG.1AV1 rats.

MATERIALS AND METHODS

Rats

Inbred DA, LEW.1AV1 and PVG.1AV1 rats were derived originally from Zentralinstitut für Versuchstierzucht, Hanover, Germany. The genetics and characteristics of the rat strains used are described in Greenhouse et al.[13]. Rats were bred, kept and used under specific pathogen-free conditions at the Centre for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden. They were maintained in climate-controlled environment with 12-h light/dark cycles, housed in polystyrene cages containing wood shavings and had free access to standard rodent food and water. The rats were sex-and age-matched for each experiment. Experimental procedures involving animals were performed according to guidelines provided by the central board for animal experiments at the Swedish Department of Agriculture, and were approved by the Ethical Board for animal experiments in Stockholm-North.

Induction and evaluation of squalene-induced arthritis (SIA)

Arthritis was induced under anaesthesia by an intradermal injection at the base of the tail with 200–220 μl squalene (C30H50, Sigma Chemicals, St Louis, MO, USA). The tritium-labelled squalene was purchased as a mix with hexane:ethanol (NEN Chemicals, Boston, MA, USA) which was evaporated by a surplus flow of nitrogen gas for 1 h. The tritium-labelled squalene was thereafter diluted with cold squalene to yield an injected tritium-dose of 0·8 μCi per rat. Arthritis development was monitored using a macroscopic scoring system ranging from 0 to 4 for each of the four limbs (1, enlargement of one type of joint; 2, enlargement of two types of joints; 3, more than two types of joints involved and 4, severe arthritis in the whole paw), yielding a score of 0–16 per animal.

T cell depletion

Monoclonal antibodies (MoAbs), purified from hybridoma supernatants using standard protein G affinity chromatography, were solved in phosphate-buffered saline (PBS) and intraperitoneally injected into DA rats at a dose of 1 mg/rat, 24 h prior to adjuvant injection. The MoAb used were directed against αβ-TcR (R73, complete depletion at 0·08 mg/rat) [14,15] and TNP (D10, used as isotype-matched, negative control). The hybridomas were kindly provided by Dr Tomas Hünig, Würzburg, Germany (R73) and Dr Birgitta Heymann, Uppsala, Sweden (D10).

Dissection of organs

Rats were sacrificed and blood samples – of which plasma samples were collected – were taken. Organs were dissected out in the following order: axillary lymph nodes, thymus, heart (rinsed in PBS to remove surplus blood), bronchiolo-alveolar package (rinsed in PBS), salivary glands, inguinal LN (of which half were used for sectioning and half for scintillation counting), liver (1/6 was dissected out for scintillation counting), spleen (of which half was used for sectioning and half for scintillation counting), left kidney, adipose tissue, stomach, intestines, m. quadriceps femoris biopsy, spinal cord, brain, hind paws (left paw was used for sectioning and right paw was used for scintillation counting) and the injection site at the base of the tail. The organs were weighed and stored at − 70°C until analysis.

Organ dissolving and scintillation counting

Organs were dissolved in Opti-Solve (Wallac, Turku, Finland), 1 ml Opti-Solve per 100 mg tissue, according to the purchaser’s protocol, diluted with Opti-Phase Hi-Safe 3 (Wallac) and put into a β-scintillation counter (Wallac 1215 Rack β II). Each organ was measured in duplicate and each sample was measured for 10min. The total decay per minute (dpm) for each organ was corrected due to background dpm. When organs were dissolved in more than 2 ml Opti-Solve, the mean dpm was multiplied with the total ml Opti-Solve used. Similarly, if less than a whole organ was put into Opti-Solve, the dpm was corrected. Since some Opti-Solve evaporated during the dissolving phase, evaporation corrections were made. Since the total dpm differed between rats, we compared the relative amounts of dpm (% of total dpm in individuals) detected in each organ. The concentration of tritium-labelled squalene in each organ (dpm/g tissue) was compared in absolute numbers.

Separation of cells and extracellular matrix

LN cell suspensions were prepared by passing the LN through a stainless-steel mesh. The cell suspensions were centrifuged into preweighed tubes and the cell pellet was weighed and stored at −70° until dissolving. The extracellular matrix stuck in the stainless-steel mesh was collected, weighed and stored at −70° until dissolving.

Adoptive transfer of arthritis

Mitogen-activated cells were prepared as follows: single cell suspensions were prepared. LN cells from several animals were pooled and cultured at 1 × 107 cells/ml with 3 μg/ml concanavalin A (con A) in Dulbecco’s modified Eagle’s medium supplemented with glutamine, streptomycin, d-penicillin, HEPES, β-mercaptoethanol and 10% fetal calf serum. The cells were cultured at 37° in a humidified 5% CO2 atmosphere for 48 h.

Naive DA rats were gamma-irradiated with 6 Gray (Gy) from a 60Co-source and 24 h later injected intravenously with mitogen-activated LN cells, either 0·25–1 × 108 axillary plus brachial LN cells or 1 × 108 inguinal LN cells. The mitogen-activated LN cells were washed three times in sterile PBS, resuspended in 1 ml cold PBS and kept on ice prior to transfer.

Statistical methods

Student’s t-test and non-parametric two-tailed ranking tests (Mann–Whitney and Kruskall–Wallis) were used as indicated in the text. P-values below 0·05 were considered significant. The statistical program used was StatView 5·0 (SAS Institute Inc., Cary, NC, USA).

RESULTS

Distribution of tritiated squalene in rats at different time-points

Nine DA rats injected with [3H]squalene were sacrificed at days 5, 10 and 15 postinjection (p.i., three per time-point). Of these, only the animals at day 15 p.i. had clinical signs of arthritis (arthritis scores of 9, 3 and 3). When the organs/tissues from all rats were analysed for tritium content, several observations were evident. First, a minor amount of squalene was deposited in the peripheral joints, i.e. hind paws (<1% of the total dpm measured, all time-points). Secondly, the major part of squalene was detected at the site of injection (42–94%, median values of 83, 93 and 71% for rats taken at days 5, 10 and 15 p.i, respectively). Thirdly, except for the injection site and the liver, the main site of deposition was in the inguinal LN draining the injection site (median values of 1, 3 and 10% of the total dpm for the three time-points, respectively), but not in the non-draining axillary LNs (all individual values were below 1%). The increased deposition in the inguinal LNs was noted from day 5 p.i. to day 15 p.i. (P =·0495, Mann–Whitney ranking test, at all three time-points). The distribution data for each individual rat at all time-points are summarized in Table 1.

Table 1
Distribution of squalene in DA rat organs taken at different time points postinjection presented as proportional distribution (% of total dpm for each individual rat)

Concentration of tritiated squalene at different time-points

The organs dissected out were weighed and their tritium contents were calculated as a concentration of dpm/g tissue (Table 2) and are here presented as median values per time-point. The injection site (median concentration 5, 10 and 15 days p.i. 711 600; 627 600; and 617 000dpm/g, respectively) and inguinal LNs (50 300, 80 700 and 100 100dpm/g, respectively) were more filled with tritiated squalene than the other tissues (median concentration 5, 10 and 15 days p.i. 2000, 1500 and 2100 dpm/g, respectively). The affected paws, on the other hand, had a very low concentration of squalene (median concentration 5, 10 and 15 days p.i. 390, 350, and 360dpm/g, respectively). Another observation was that the concentration in non-draining axillary LNs (1500, 600 and 1600dpm/g, respectively) compared to draining inguinal LNs was considerably lower (P =0·0043, paired t-test, at each of three time-points). Although a large amount of squalene was deposited in the liver and gastrointestinal tract (mean percentages of 11·9 and 1·7, respectively, of the total dpm measured), the concentrations in these organs (mean concentrations, all individuals, of 6500 and 900dpm/g tissue, respectively) were substantially lower than in the inguinal LNs.

Table 2
Concentration of 3H-labelled squalene in organs of DA rats from different time points postinjection, presented as dpm/g tissue

Squalene deposition in different rat strains and in T cell depleted DA rats

A comparison of squalene deposition over time in arthritis-susceptible DA rats and arthritis-resistant but MHC-identical LEW.1AV1 and PVG.1AV1 rats revealed a similar adjuvant distribution in all strains (n = 3/time-point and strain, data not shown). Overall, a high deposition of oil in the injection site (79% of total dpm in DA rats, 89% of total dpm in LEW.1AV1 rats and 85% in PVG rats, respectively) and in the draining inguinal LNs (4·3%, 6·1% and 2·2%, respectively) was observed, with a very low deposition in the hind paws (0·2%, 0·1% and 1·0%, respectively). In order to determine whether T cells affect the transport of oil from the site of injection, a T cell-depletion was performed in four DA rats and compared to three DA rats receiving irrelevant MoAbs. No differences in squalene deposition or concentration was noted between normal rats and T cell-depleted rats (data not shown). All rats displayed a similar distribution; very low amounts of tritium were detected in the non-draining LNs (median dpm/g tissue in non-draining control LNs was 1200 compared to 2200dpmsol;g tissue in T cell depleted rats) whereas the draining LNs were heavily loaded with squalene (median dpm/g tissue in draining control LNs was 45 600 compared to 28 900dpm/g tissue in the T cell-depleted rats).

Hyperplasia in lymph nodes

The LNs dissected out from DA and LEW.1AV1 rats at days 5, 10 and 15 postinjection were compared to naive, normal rats with respect to weight, as an estimate for hyperplasia. Inguinal LNs from DA rats had increased in weight at days 5, 10 and 15 postinjection compared to normal rats (median weight day 5, 10 and 15 p.i., 0·116, 0·176 and 0·222 g, compared to normal weight of 0·040 g; P =0·03, 0·0003 and 0·0012, respectively, unpaired t-test). The axillary LNs from DA rats were also hyperplastic, as reflected in an increase in weight at day 15 compared to normal axillary LNs (median weight: 0·076g versus 0·042 g; P =0·0009, unpaired t-test). Inguinal LNs taken from LEW.1AV1 rats were similarly increased in weight at days 5, 10 and 15 after squalene injection compared to normal LEW.1AV1 rats (median weight days 5, 10 and 15 p.i., 0·160, 0·174 and 0·232 g, compared to normal weight of 0·038 g; P < 0·001, P < 0·001 and P =0·009, respectively, unpaired t-test), and axillary LNs showed a weight increase at days 5 and 10 p.i. compared to normal axillary LNs (median weights: 0·069 and 0·057g versus 0·038 g; P =0·015 and P =0·025, respectively, unpaired t-test). Inguinal LN from PVG.1AV1 rats were also hyperplastic but less so than LN from both DA and LEW.1AV1 rats at day 10 p.i. and from DA rats at day 15 p.i. (median PVG.1AV1 LN weights of 0·035 and 0·083g at days 10 and 15 p.i., respectively).

Squalene deposition in lymph node cells and extracellular matrix

To determine the location of squalene in DA LNs, a separation of cells and extracellular matrix (ECM) was performed. When comparing cells from different LN, most of the tritium-labelled oil was detected in the inguinal LN cell suspension (90% of all dpms) and only small amounts in other LN cell suspensions (0·9%, Table 3). The squalene content in the inguinal LN cells (4800dpm) was 240 times higher than in cells from the axillary LNs (20dpm) and 160 times higher than the brachial lymph node cells (30dpm), respectively. The ECM contained small amounts (9% in total), of which the ECM derived from inguinal LNs was the major contributor (7·1%).

Table 3
Distribution of 3H-labelled squalene in lymph node cells and extracellular matrix (ECM), respectively, from lymph nodes of two pooled DA rats, taken at day 5 postinjection

Adoptive transfer of squalene-induced arthritis

Cells from either inguinal LNs or axillary and brachial LNs were transferred from arthritic DA rats to naive, sex-matched DA rats, and the recipients were clinically evaluated for arthritis (Table 4). Cells from the draining inguinal LNs were transferred with a 100% incidence. A lower but substantial arthritis incidence, 50%, was recorded in recipients receiving non-draining axillary/brachial LN cells. In the experiments, a higher incidence was achieved in males (100%) compared to females (60%) Arthritis severity scores and day of onset did not, however, differ between sexes or between lymph node cell origins.

Table 4
Arthritis clinical data from four pooled cell transfer experiments in DA rats, using different lymph node cell origins

DISCUSSION

Delineating the pathways by which adjuvants can induce arthritis in rats is important for the understanding of this RA-like disease. Here, we address the intriguing question of how adjuvants induce joint-specific inflammation. So far, there is no evidence that autoimmunity could account for joint restriction in SIA, or in any other model induced by non-specific immunostimulation [9,10,12,16]. Another possibility, which is the main focus of this study, is that the unique joint architecture leads to concentration of adjuvants in peripheral joints, hence focusing adjuvant-induced inflammatory reactions to this site [10]. A previous report showed that arthritogenic 14C-labelled hexadecane disseminates into several organs, but most prominently into LN [17]. Peripheral joints, however, were excluded from that investigation.

Here it is demonstrated conclusively that squalene does not accumulate in peripheral joints. We show that the squalene concentration in these joints is lower than in most organs, including the brain and spinal cord. Yet, adjuvant-injected DA rats develop arthritis rather than encephalomyelitis, although they are highly susceptible in models for both RA and multiple sclerosis [5,9,10,1821,. Admittedly, we cannot exclude that adjuvants may concentrate in a minor tissue-or cell-type present in the joints. This possibility is technically difficult to investigate, but it could be achieved by autoradiography of [3H]labelled squalene, since the wavelength of emitted radiation enables resolution well below cell size. Nevertheless, the present study on adjuvant spreading suggests strongly that transportation to joints is not the explanation for joint-specific inflammation.

Our data rather propose that critical activation events take place in LN. Thus, we present original data that LNs become hyperplastic, as evidenced by a large increase in size following adjuvant injection. In addition, LN cells become arthritogenic, since they are capable of transferring SIA after conA-stimulation and injection into irradiated recipients. Notably, the described activation events were not closely linked to concentration of adjuvant in LNs. Although squalene accumulated preferentially in LNs draining the injection site rather than other LNs (>300-fold difference in concentration), lymphoplasia and arthritogenic cells are present in both types of lymph nodes.

Importantly, the mere presence of adjuvant in LNs is not sufficient for arthritis induction, since a similar distribution of squalene was observed in DA rats and in arthritis-resistant but MHC-identical LEW.1AV1 and PVG.1AV1 rats. These rats also developed LN hyperplasia, although to different degrees, suggesting that they are not inert to the adjuvant exposure, but that their resistances lie in adjuvant responses rather than distribution of adjuvant. Neither were T cells found to be important for squalene distribution, since abrogation of arthritis by administrating αβ T cell MoAbs prior to adjuvant injection did not change the distribution of squalene.

Our results demonstrate that [3H]labelled squalene preferentially accumulates in draining LNs, which differs from another report showing accumulation in all LNs for 14C-labelled hexadecane [17]. This discrepancy could reflect that the previous study was not focused on a precise LN comparison, or that the methodology used was only semiquantitative. In our opinion, the difference may also be biological, i.e. the two arthritogenic adjuvants could have different dissemination patterns, depending on their biological structure and origin. These dissemination patterns are important to delineate, since they can give information on where the immune system encounters arthritogenic adjuvant.

It remains to be determined how adjuvant oils are transported in vivo. They could be transported as droplets, as micelles or in association with certain lipid-transporting molecules. A more likely explanation is that they are transported by cells, for example by macrophages and/or dendritic cells moving from the site of injection to draining LNs [22]. Our data showing squalene being present in LN cells support the cellular transport hypothesis. Adjuvants could penetrate into cells or cell membranes where they may change membrane fluidity and modulate transcriptional regulation [23]. It is also possible that the lipid squalene could bind to the CD1 molecule and, thus, be recognized by CD1-restricted T cells [2426], but there is no evidence to support this hypothesis. In fact, squalene is a weak antigen that is normally non-immunogenic [27], and it is highly unlikely that the squalene preparation contains arthritogenic proteins [10]. How then does this lipid cause arthritis?

It has been demonstrated previously that the adjuvant oil squalene [28,29] induces a systemic response in rats, such as increased blood levels of acute phase proteins [10]. It is therefore possible that components of this response affect joints, which then selectively attract T cells activated by adjuvants deposited in the LNs. These lymphocytes may have no particular specificity, or may be specific for endogenous antigens expressed under stressful conditions, such as heat shock proteins. They may also be specific for joint-derived antigens. The failure hitherto to demonstrate such autoimmunity could lie in the types of responses tested, or in the types of autoantigens tested.

At present, the pathways leading from adjuvant injection to arthritis remain elusive [30], although models induced by non-immunogenic adjuvants were first described more than 20 years ago [6,31,32]. A possible delineation of disease pathways may come from genetic dissections of adjuvant arthritis models. So far, genome-wide scans have identified several chromosome regions that are linked to arthritis induced with adjuvant oils [3335]. Two of these quantitative trait loci were linked to arthritis induced by incomplete Freund’s adjuvant (IFA) and squalene [10,33]. Both of these quantitative trait loci are now reproduced in congenic rat strains [36,37], and the corresponding human chromosome regions (12p and 17q) have been linked to rheumatoid arthritis in independent studies [3840]. Therefore, we believe that genetic insight into disease pathways can be expected in rats, and possibly in humans.

Acknowledgments

The authors would like to thank Åsa Jansson PhD for purification of antibodies. We are also thankful to Vivianne Malmström PhD for critical reading of the manuscript. This study was supported by grants from the Swedish Medical Research Council, King Gustav V’s 80th Birthday Jubilee Foundation, the Åke Wibergs Foundation, the Ulla and Gustaf af Ugglas Foundation, the Alex and Eva Wallström Foundation, the Nanna Svartz’ Foundation, Ålands Lagting and from the Swedish Rheumatism Association. J.C.L. is in receipt of a Fellowship from the Swedish Medical Research Council.

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