Resistance to re-challenge in the Brown Norway rat model of vasculitis is not always complete and may reveal separate effector and regulatory populations

Abstract

Administration of mercuric chloride to Brown Norway rats results in T helper type 2 (Th2)- dominated autoimmunity characterized by high immunoglobulin E (IgE) concentrations, the production of multiple IgG autoantibodies, including those to glomerular basement membrane (GBM), arthritis and caecal vasculitis. After 14 days animals immunoregulate and auto-immunity resolves even if mercuric chloride injections are continued. In a third phase, if animals are re-challenged with mercuric chloride 6 weeks later, they show only attenuated autoimmunity with lower anti-GBM antibody concentrations and arthritis scores. Resistance to the induction of anti-GBM antibodies can also be achieved following an initial challenge with low-dose (one-tenth standard dose) mercuric chloride. We have now studied this resistant phase in more detail. We have shown, first, that following an initial full-dose mercuric chloride challenge, resistance also affects susceptibility to caecal vasculitis. Second, following an initial full-dose mercuric chloride challenge, the IgE response upon re-challenge is initially accelerated but subsequently enters a resistant phase and third, following an initial challenge with low-dose mercuric chloride, resistance is also seen to the induction of caecal vasculitis but is not seen in IgE serology (where results suggest competing effector and regulatory cell populations). Studying such regulatory phases in animal models of autoimmunity may be of benefit in the future in designing new therapies for human vasculitis.

Introduction

The human vasculitides are autoimmune diseases characterized by inflammation and necrosis of blood vessels resulting in vascular occlusion and tissue ischaemia.¹ They present as multisystem disorders affecting organs such as the kidney and brain and, if left untreated, they may be fatal. Characteristically vasculitis shows a relapsing and remitting course, where, following a period of disease activity, patients may spontaneously pass into a remission phase where they appear refractory to the induction of further autoimmunity. Current treatments for vasculitis usually rely upon non-specific immunosuppression with associated risks of infection, infertility and malignancy. If, instead of interrupting the pathogenesis of disease, we could mimic the immunoregulatory events that lead to spontaneous remission, we might be able to develop less toxic forms of therapy.

The administration of mercuric chloride (HgCl₂) to Brown Norway rats results in a T helper type 2 (Th2) -dominated autoimmune syndrome characterized by high immunoglobulin E (IgE) concentrations,² production of multiple IgG autoantibodies including those to glomerular basement membrane (GBM) and collagen,^3–6 distal polyarthritis,⁷ caecal vasculitis,⁸ severe mucositis and significant weight loss. Disease peaks approximately 14 days after the start of HgCl₂ injections, after which autoimmunity spontaneously improves even if HgCl₂ injections are continued.^2,3,7,8 In a third separate phase if animals are re-challenged with HgCl₂ between 1 and 4 months after their original challenge, they develop only attenuated autoimmunity with reduced concentrations of anti-GBM antibodies⁹ and little arthritis.⁷ The severity of caecal vasculitis and the course of the IgE serological response have never been studied in this resistant phase.

Resistance in this model has also been demonstrated using a lower dose of HgCl₂. Previous studies have shown that if animals are initially challenged with one-tenth of the standard dose of HgCl₂, instead of developing full-blown autoimmunity, they develop only mildly raised anti-GBM antibody concentrations, with no data available on other aspects of the model.¹⁰ On repeat challenge with full-dose HgCl₂ 6 weeks later, however, these animals also develop only attenuated levels of anti-GBM antibodies, suggesting that the initial challenge, whilst resulting in only modest stimulation of effector cells, has, none the less, activated a regulatory population.¹⁰ Whether low-dose initial challenge results in resistance to the induction of caecal vasculitis or to the induction of the IgE serological response has, once again, never been studied.

Although animals models rarely reflect human disease directly, the description in the Brown Norway rat model of widespread arthritis and a gut vasculitis in the context of raised IgE concentrations is reminiscent of the human disease Churg–Strauss syndrome, although the marked eosinophilia seen in this condition is not reflected in the experimental model (D.B.G. Oliveira, unpublished observations). Of particular interest is the time course of the model apparently passing through relapsing and remitting phases. By understanding the immunoregulatory events that carry animals into the resistant phase of this model, we may gain insights which in the future will help us to design more specific therapies for human vasculitis.

Studying which aspects of autoimmunity enter a resistant phase is not of purely descriptive value but may also help us to dissect the mechanism of resistance in this model. Although arthritis, vasculitis and serological changes are all known to be αβ T-cell dependent,^7,11 with the large rise in IgE concentrations implicating the Th2 cell (following an interleukin-4 driven IgE class switch), there are also clear differences in the pathogenesis of different aspects of the syndrome. Caecal vasculitis is unique in being partly neutrophil dependent¹² whilst the severity of arthritis alone demonstrates a CD8 dependence.¹³ With both shared and unique aspects to pathogenesis, we would predict that if the mechanism of resistance were acting at a proximal level of pathogenesis, then resistance would be broad, affecting all aspects of the syndrome. If, however, the mechanism of resistance was targeted on downstream unshared events, then differential regulation would be seen. We therefore chose to investigate in more detail the resistance seen in this model following an initial challenge with both full and low-dose HgCl₂.

Materials and methods

Induction of the animal model of vasculitis

Experimental protocols for individual experiments are shown in Table 1.

Table 1.

Individual experimental protocols

	Experiment	Mean group weight at start of challenge (g)	First challenge	Second challenge	Re-challenge day	Bleed days after start of second challenge	Kill day after start of second challenge
1	Control (n = 5)	222 ± 34·4	Saline	HgCl2	43	1, 9, 14, 19, 22 and 26	26
	Test (n = 6)	212·5 ± 14·4	HgCl2	HgCl2
2	Control (n = 7)	283·6 ± 9·4	Saline	HgCl2	43	1, 7 and 15	15
	Test (n = 6)	282·8 ± 25·2	HgCl2	HgCl2
3	Control (n = 6)	290 ± 9·9	Saline	HgCl2	42	1 and 3	3
	Test (n = 7)	263 ± 31·5	HgCl2	HgCl2
4	Control (n = 5)	311·4 ± 21·1	Saline	HgCl2	43	1 and 5	5
	Test (n = 9)	274·5 ± 18·5	HgCl2	HgCl2
5	Control (n = 10)	261·7 ± 14·1	Saline	HgCl2	42	1, 5, 8, 11 and 14	14
	Test (n = 10)	246·9 ± 24·9	Low-dose HgCl2	HgCl2

Male Brown Norway rats (150–400 g) were obtained from Harlan Olac (Bicester, UK), given food and water ad libitum and were used in age-matched groups. All experimental procedures were performed under halothane anaesthesia.

Autoimmunity was induced using five subcutaneous injections of 1 mg/kg of a 0·1% solution of HgCl₂ between days 1 and 8. In experiment 5 this first HgCl₂ challenge was performed using low-dose HgCl₂ (0·1 mg/kg of a 0·1% solution of HgCl₂). Control animals were given an equivalent volume of normal saline. A second challenge was performed using between one and five injections of full-strength HgCl₂ beginning 41 or 42 days after the start of the first HgCl₂ challenge. In experiments 3 and 4, where animals were killed at 48 and 93 hr after the start of their second challenge, they had received only either one (experiment 3) or three (experiment 4) injections of HgCl₂ before they were killed. Animals were weighed regularly throughout the experiment and serial blood samples were taken after each challenge by tail artery puncture. In each experiment the time between the start of the first HgCl₂ or saline challenge and the day before the start of the second challenge is designated phase 1 and the time between the start of the second HgCl₂ challenge and the end of the experiment is designated phase 2. Following repeat challenge with full-dose HgCl₂ animals often fail to tolerate the repeated anaesthesia required for sequential blood sampling. For this reason information about the full time course of IgE serology following repeat full-dose HgCl₂ re-challenge has been derived from several different experiments (experiments 1, 2, 3 and 4) each sampling IgE concentrations at different time-points after the second challenge. In experiment 1 the control and test animals were housed together in the same cages. In all other experiments control and test animals were housed in separate cages.

Vasculitis scores

Macroscopic caecal vasculitis was scored at necropsy on day 14 or 15 by an experienced observer (D.B.G.O.) blinded to treatment using a previously validated scoring system.¹⁴ Both serosal and mucosal surfaces were assigned a score between 0 and 4 which were combined to give a final score. Directed caecal biopsies were then stained with haematoxylin & eosin and were examined by an experienced histopathologist (D.R.T.) to give a microscopic caecal vasculitis score on a scale of 0–4 as previously described.¹⁵

Arthritis score

Daily arthritis scores of wrist and ankle joints were recorded by an experienced observer (C.S.V.) blinded to treatment on the following scale:⁷ 1, erythema only; 2, swelling confined to the wrist or ankle joint; 3, swelling extending distally on to the fore or hind foot; 4, gross swelling involving the whole of the fore- or hind-foot. Scores are expressed either as the total daily scores for individual animals or as the cumulative total arthritis score for an animal on a number of days between days 12 and 18.

Serology

Total IgE concentrations and levels of anti-collagen antibodies were measured using standard enzyme-linked immunosorbent assay technology.⁷

Statistical analysis

Statistical analysis was performed using the instat 2 version 2·04a computer program (Graph Pad software) and the windows spss 11 program (used for repeated measures analysis of variance calculations). Caecal vasculitis scores were compared using the Mann–Whitney U-test. IgE concentrations were log-transformed to base 10 and were usually compared using repeated measures analysis of variance to give two P values; the first is an analysis of the group effect whilst the second is an analysis of the interaction effect between group and time. Where analysis compared only the increment in IgE concentration between two time-points Mann–Whitney U was again used. In all cases a two-tailed P-value < 0·05 was considered to be significant.

Results

Results seen after a full-dose HgCl₂ re-challenge following a full-dose HgCl₂ pre-challenge

IgE concentrations.

In experiment 1 (Fig. 1), following a full-dose HgCl₂ first challenge, animals re-challenged with HgCl₂ showed significantly lower IgE concentrations than after their first challenge (anova group effect P < 0·001, interaction effect P = 0·001) although peak concentrations were still at day 14 and were followed by spontaneous regulation. In this first experiment the control and test animals were housed in the same cages. Animals re-challenged with HgCl₂ at day 43 did not show significantly lower IgE concentrations than control animals challenged with mercuric chloride for the first time at day 43, who showed unusually low IgE concentrations. A similar picture was seen for IgG anti-collagen antibody serology (data not shown).

Figure 1.

Experiment 1: IgE concentrations after first- and second-challenge full-dose HgCl₂ challenge in control (first-challenge saline, second challenge HgCl₂) and test (both first and second challenges HgCl₂) animals. ANOVA comparing IgE concentration after first and second challenge in test animals group P < 0·001, interaction P = 0·001.

In experiment 2 (Fig. 2), where test and control animals were housed in separate cages, test animals challenged with HgCl₂ for a second time showed a significantly lower overall rise in IgE concentration between day 1 and day 15 of re-challenge than control animals receiving their first challenge with HgCl₂ (Mann–Whitney U, P = 0·001). However, as shown below, on more detailed analysis of early time-points it is clear that the immediate response of the test group in the first 93 hr after re-challenge is actually accelerated compared to that seen in the control group.

Figure 2.

Experiment 2: IgE concentrations after first and second full-dose HgCl₂ challenge in control (saline/HgCl₂) and test (HgCl₂/HgCl₂) animals. Mann–Whitney U-test, comparing increment in IgE concentrations after second challenge in control and test animals, P = 0·001.

Experiments 3 and 4 were designed to study the IgE response in animals at early time-points following an HgCl₂ re-challenge. The increment in IgE concentration between the start of the second challenge to that seen 48 hr (experiment 3) and 93 hr (experiment 4) later was significantly higher in the animals re-challenged with HgCl₂ for the second time than in the animals challenged with HgCl₂ for the first time (Fig. 3) (Mann–Whitney U, P = 0·0012 for experiment 3 and P = 0·001 for experiment 4).

Figure 3.

Experiments 3 and 4: early time course of IgE concentrations after full-dose HgCl₂ re-challenge as seen in control (saline/HgCl₂) and test (HgCl₂/HgCl₂) animals in experiment 3(a) and 4(b). Mann–Whitney U-test, P = 0·0012 comparing test and control group IgE increments after second challenge.

Caecal vasculitis

In experiment 2 (Fig. 4), following a full-dose HgCl₂ initial challenge, animals re-challenged with HgCl₂ showed significantly lower macroscopic and microscopic caecal vasculitis scores than those seen in animals challenged with HgCl₂ for the first time [Mann–Whitney U; P = 0·0023 (macroscopic caecal vasculitis) and P = 0·0012 (microscopic caecal vasculitis)].

Figure 4.

Experiment 2: Macroscopic (a) and microscopic (b) caecal vasculitis scores seen after a second full-dose HgCl₂ challenge in control (saline/HgCl₂) and test (HgCl₂/HgCl₂) animals. Mann–Whitney U-test, P = 0·0023.

Results following a first challenge with low-dose HgCl₂

IgE concentrations

In experiment 5 after a first low-dose HgCl₂ challenge test animals showed a three-log-fold IgE concentration rise compared to the control animals (Fig. 5; phase 1). As with a full-dose HgCl₂ challenge, the peak concentration was seen at approximately day 14 after the start of HgCl₂ injections and was followed by spontaneous autoregulation (anova group and interaction effects P < 0·001 comparing control and test groups).

Figure 5.

Experiment 5: IgE concentrations seen after a first challenge with low-dose HgCl₂ (phase 1) and a second challenge with full-dose HgCl₂ (phase 2) in control (saline/full-dose HgCl₂) and test (low-dose HgCl₂/full-dose HgCl₂) animals.

Arthritis

In experiment 5 after a first low-dose HgCl₂ challenge, five of the 10 test animals developed arthritis beginning on day 14 after the start of HgCl₂ injections (Fig. 6) whilst none of the control group developed arthritis. The severity of the arthritis was not diminishing on day 18 when observations were stopped. We have observed this later time course of arthritis following low-dose HgCl₂ challenge on several occasions (data not shown).

Figure 6.

Experiment 5: mean daily arthritis scores in test animals after a low-dose HgCl₂ challenge.

Results after a full-dose HgCl₂ re-challenge following a low-dose HgCl₂ first challenge

IgE concentrations

In experiment 5 (Fig. 5; phase 2) following a low-dose HgCl₂ pre-challenge, animals challenged with full-dose HgCl₂ showed an accelerated rise in IgE concentration between days 1 and 5 of re-challenge and then went on to show day 8, 11 and 14 re-challenge values which oscillated around those seen after a first challenge in the control group (anova group and interaction effects comparing control and test groups P < 0·001). However, the time course of IgE concentrations for control and test groups was markedly different. Control animals receiving their first HgCl₂ challenge showed a consistent rise in IgE concentration throughout the 14 days of the second challenge whereas animals in the test group demonstrated alternating increments or decreases in IgE concentrations with each successive time-point, with the effect seen both as a group and in individual animals (Fig. 7). A similar picture was seen in IgG anti-collagen antibody serology (data not shown).

Figure 7.

Experiment 5: comparison of IgE concentrations in individual control [saline/full-dose HgCl₂ (a)] and test [low-dose HgCl₂/full-dose HgCl₂ (b)] animals during their second HgCl₂ challenge. (Detailed comparison of data previously shown in summary form for phase 2 in Figure 5.)

Caecal vasculitis

In experiment 5 following low-dose HgCl₂ first challenge, animals re-challenged with full-dose HgCl₂ showed significantly lower macroscopic and microscopic caecal vasculitis scores than animals receiving their first HgCl₂ challenge (Fig. 8– Mann–Whitney U macroscopic vasculitis, P = 0·028, microscopic vasculitis, P < 0·001).

Figure 8.

Experiment 5: macroscopic (a) and microscopic (b) caecal vasculitis scores seen after rechallenge with full-dose HgCl₂ after an initial challenge with low-dose HgCl₂ in control (saline/full-dose HgCl₂) and test (low-dose HgCl₂/full-dose HgCl₂) animals. Mann–Whitney U-test, P = 0·028.

Discussion

We have shown for the first time that if animals are challenged for a second time with full-dose HgCl₂, they show broad resistance affecting caecal vasculitis and IgE concentrations as well as arthritis and anti-GBM serology as already described. As a result of the fragile state of the animals, multiple bleeds are often not tolerated – we have therefore constructed our time course from the results of several different experiments. Another important limitation of our results is that because of the fragile state of the animals referred to above, it has not been possible to repeat the re-challenge experiments; our conclusions should therefore be viewed with suitable caution.

In experiment 1 after a second full-dose challenge, although day 9 IgE concentrations were not significantly different between HgCl₂ challenges, day 14 IgE concentrations were significantly lower at re-challenge than at first challenge. The time course of IgE serology showed a similar picture in both challenges, with concentrations peaking at approximately day 14 and then autoregulating. However, more detailed studies on the first 93 hr of the second challenge (experiments 3 and 4) have shown that resistance is not immediately effective, with higher IgE values seen in the test than in the control group. Results show the response to be initially accelerated and only later controlled by a regulatory cell, a situation reminiscent of that seen following re-challenge in the rat erythrocyte-induced anti-erythrocyte autoantibody model of autoimmunity.¹⁶ The initially accelerated response implies that full activation of the regulatory population may require re-stimulation with HgCl₂, an observation consistent with previous experiments showing that resistance to re-challenge with HgCl₂ wanes with time.⁹

The results in control animals in experiment 1 were unexpected, with animals thought to be challenged for a first time also showing lower IgE concentrations than those seen in the test group after a first full-dose HgCl₂ challenge. We believe that whilst the test group phase 2 results reflect true resistance at re-challenge, results from control animals are abnormal because of the experimental conditions. In experiment 1 control and test animals were housed in the same cages and we speculate that aerosol inhalation, licking of HgCl₂ injection sites and coprophagia may have led to ‘control’ animals receiving a small phase 1 HgCl₂ challenge. Such a dose, whilst not sufficient to engage an effector population, may have been able to stimulate a regulatory population causing animals to demonstrate later resistance. In support of this, we note that in experiment 2 where control and test animals were caged separately resistance was seen only in the test group as shown by caecal vasculitis scores, and day 15 IgE concentrations.

Results from experiment 5, where animals received a low-dose HgCl₂ first challenge, are interesting, being different from those of Bowman et al.¹⁰ who showed only very modest levels of anti-GBM antibodies¹⁰ following low-dose HgCl₂ with subsequent complete resistance to re-challenge with full-dose HgCl₂ as measured by the anti-GBM antibody response. Studies by Szeto et al.¹⁷ on low-dose HgCl₂ challenge showed IgE concentrations three-log-fold lower than those seen after a full-dose challenge followed by incomplete resistance on full-dose rechallenge with rising levels of IgE after day 20. The cytokine environment of the initial response to HgCl₂ was also dose-dependent with a Th1 bias and a progressive rise in interferon-γ levels following low-dose challenge compared to the normal Th2 bias and up-regulation in interleukin-4 mRNA seen after full-dose HgCl₂.¹⁸ By contrast in our studies, following low-dose challenge, animals showed significantly elevated concentrations of IgE with a three-log-fold rise in concentration.

Neither previous group studied the incidence of arthritis following an initial low-dose challenge. In earlier studies following full-dose HgCl₂ challenge⁷ arthritis was seen in more than 80% of animals (100% of male rats) beginning at day 12, peaking at day 15 and then improving. In our studies arthritis was seen in 50% of animals but was consistently delayed compared to that seen after a full-dose challenge beginning at day 14 and still worsening in severity as observations stopped at day 18.

We did not measure anti-GBM antibodies in our animals and therefore direct comparison with some previous results from other groups is not possible; however, our experience is that the anti-collagen and anti-GBM antibodies behave in a similar fashion. Discrepancies between the groups in serological results may reflect differences in genetics between animals used by the groups or differences in experimental conditions, such as the gut pinworm load which is known to affect serological read-outs and to make comparison of results between laboratories difficult. Differences in results seen after low-dose HgCl₂ challenge are also easier to understand if we consider the number of different cell populations affecting the magnitude and duration of the first challenge response. HgCl₂ not only stimulates an αβ⁺ CD4⁺ T-cell effector population,¹¹ resulting in serological changes and tissue damage, but also results in a diminution of CD8⁺ cell numbers between days 5 and 10, followed by a recovery in CD8⁺ cell numbers during the autoregulatory phase of the model, suggesting that autoimmunity might in part be the result of inhibition of a CD8⁺‘suppressor’ population that normally prevents autoimmunity.^19,20 Other cells also act as a check to the effects of HgCl₂, with studies showing that the use of cyclophosphamide (thought to differentially inhibit suppressor T-cell function) can increase the magnitude of the day 14 serological response to HgCl₂, suggesting that such a cell normally limits this response.⁹ Using limiting dilution analysis, Rossert et al.²¹ have also shown the presence in vitro of a CD8⁺ cell capable of suppressing effector cells in this model derived from day 14 animals. With many elements combining to determine the first-challenge serological response, even small differences in the dosing regimes between the three studies may dramatically alter the final result depending on their differential effects on a particular cell subset.

In experiment 5, after a low-dose pre-challenge once again animals challenged with full-dose HgCl₂ show relative resistance to the induction of caecal vasculitis [and a trend towards resistance in arthritis scores (data not shown)]. In results similar to those seen after a full-dose HgCl₂ pre-challenge, the low-dose pre-challenge animals also show an accelerated IgE response between days 1 and 5 of re-challenge. Subsequently, however, animals do not show true resistance as measured by re-challenge phase IgE concentrations but have day 8, 11 and 14 IgE values which oscillate around those of the control group and are suggestive of alternating populations of regulatory and counter-regulatory effector cells exchanging dominance in this phase, causing the variable level of IgE concentration. We therefore speculate that low-dose challenge is able to stimulate a significant effector response in the first challenge but that it has not been sufficient to stimulate a regulatory population that is potent or numerical enough to regulate the effector response upon re-challenge.

Once again the final result of HgCl₂ challenge in the resistant phase almost certainly represents a combination of its effects on a number of different cell types. In experiment 5 we appear to have differentially stimulated an effector population without fully engaging the regulatory population. Certainly examples of the differential stimulation of a regulatory population without an effector population can be found in both the Szeto et al.¹⁷ low-dose model and separately following neonatal challenge with HgCl₂ where animals do not develop autoimmunity but are rendered resistant to subsequent further challenge with HgCl₂.²² The engagement of a regulatory population without stimulation of effector cells is also seen in Lewis rats. They are resistant to the autoimmunity caused by HgCl₂ yet if challenged with HgCl₂ they show subsequent resistance to the induction of Th1-biased autoimmune diseases, such as Heymann's nephritis, to which they are normally susceptible, with resistance being mediated by a CD8⁺ regulatory cell.²³ Our own studies, by contrast, are the first to show full engagement of an effector population without full stimulation of the regulatory population with the resultant partial resistance.

The Brown Norway rat model of vasculitis is unusual as a Th2-biased model of autoimmune disease where most models of autoimmunity show a Th1 bias. The fine balance described in this paper between regulatory and effector cells and our failure to achieve true resistance in IgE serology is of particular interest because it is known that tolerance in the context of Th2 activity is more difficult to achieve than that seen in the context of Th1 activity.²⁴ We also note that whereas in most models of autoimmunity, aerosol inhalation would lead to tolerance induction, in the Brown Norway rat inhalation of HgCl₂ actually leads to autoimmunity itself²⁵ again suggesting that tolerance induction is harder to achieve in this model than in other Th1-biased autoimmune models.

In experiment 5, we have separated the manifestations of autoimmunity, inducing a regulatory population capable of controlling caecal vasculitis (and possibly arthritis) but incapable of fully regulating IgE serology. In addition, in our different experiments we have split the effector and regulatory populations. In control animals in experiment 1 we have failed to stimulate a significant effector population in phase 1 but have engaged a regulatory population subsequently revealed upon re-challenge. In experiment 2 we have stimulated a fully active effector population followed by a fully active regulatory population in the test group. In experiment 5 we have stimulated a significant effector population in phase 1 but an only partially effective regulatory population in phase 2.

Whilst the IgE serological results suggest competing regulatory and effector populations, resistance to the induction of caecal vasculitis by HgCl₂ following low-dose HgCl₂ initial challenge does appear complete. We have repeatedly observed that caecal vasculitis is more susceptible to the induction of resistance than are other aspects of the syndrome. This is consistent with the unique bias towards regulation found in the gut mucosa to protect it from inappropriate activation by harmless intestinal antigens. Cell phenotypes are specialized and different from those of the systemic immune compartment with a high proportion of CD8⁺ cells thought to be involved in regulation.^26,27 As an anatomically discrete area, with specialized lymphoid cells expressing receptors that allow their constant re-circulation to the gut-associated lymphoid tissue,²⁸ it is more feasible for a regulatory population to control autoimmunity here than in the widely distributed systemic immune department. The later time course of vasculitis beginning 10 days after the start of HgCl₂ injections may also make it more susceptible to the effects of a regulatory population which may itself require the HgCl₂ boost to become fully activated.

In summary, we have studied further the resistant phase of the Brown Norway rat model of vasculitis. After two full-dose HgCl₂ challenges resistance appears to be robust, affecting all aspects of autoimmunity. After lower dose pre-challenge, however, resistance is incomplete with data suggesting a fine balance between effector and regulatory populations. Further dissection of such systems may give useful insights into mechanisms of immunoregulation taking place during the remission phase of human vasculitis.