We have eliminated a functional gene for IL-1ra in mice and have found reproducible development of lethal inflammatory lesions at high stress sites in the major arteries. Turbulent flow causes fluctuating shear stress in vessel walls and has been suggested to alter vascular gene activation directly, leading to increased leukocyte adherence to endothelial cells 32
, and thus might result in increased leukocyte traffic through the vessel wall. Irregular flow may also allow deposition of bacterial pathogens or proinflammatory debris, including oxidized lipids, at turbulent sites. It has been shown that oxidized lipids can, for instance, activate chemokine expression in endothelium 33
. Turbulence probably also causes increased localized damage, particularly desquamation of the endothelium and local inflammation 24
. Further studies will be needed to establish whether IL-1 is expressed in mice only at sites of localized arterial damage or whether it is generally induced at sites of stress within healthy vessel walls.
In its transmural destructiveness, the disease that we have observed resembles human giant cell arteritis and polyarteritis nodosa. The former has a similar pattern of distribution within arteries but is normally characterized by frequent giant macrophages, which are rare in the animal disease. In 42 affected Il1rn−/−
animals, we have detected no active inflammation of the arteries of the kidney, which is a common feature of the latter. Human arterial disease and arteritides have been linked to microbial pathogens, and there has been recent interest in Helicobacter
spp. and Chlamydia
in coronary disease. Similarly cytomegalovirus causes arteritis in mice, although the pathological features are different from those reported here 34
. Our colony was initially established under quarantine (not SPF) conditions, but up to the time of this writing (now seven generations after rederivation into SPF conditions), Il1rn−/−
mice have continued to develop lethal arteritis. Tests for an array of viral and bacterial pathogens (see Materials and Methods) showed that all pathogens investigated were absent, with the exception of H. hepaticus
, which we have detected in both our colony and that of Hirsch et al. 27
, where vascular disease was not found. The bacterium appears to colonize littermates of all genotypes. We are, however, currently investigating the influence of the resident bacterial flora on the occurrence of arteritis.
A pattern of early mortality has emerged among Il1rn−/− animals (backcrossed onto a C57BL/6 background) from the US group (Hirsch, E., and D. Hirsh, personal communication) and illness in animals from the Japanese group, who bred their mice onto a Balb/c background (Iwakura, Y., personal communication). We have now examined specimens from the US group (backcrossed 15 times onto C57BL/6) but have not found evidence for arteritic lesions at the commonly affected sites in six Il1rn−/− specimens provided (aged 175–427 d), despite overt malaise in four of six cases. The cause of high mortality in the US Il1rn−/− animals is thus not explained.
It is noteworthy that the Il1rn−/− Balb/c mice, after the fourth backcross, spontaneously develop a quite different chronic inflammatory pathology, namely arthritis, in 80% of cases before they are 56 d old (Iwakura, Y., personal communication). We have examined bones and joints of 42 Il1rn−/− animals from our colony, all of which were older than 56 d, and have not detected arthritis, nor have we observed external signs of joint disease in the living animals.
At least a part of the cause of the difference between the three Il1rn-null colonies is likely to stem from differences in their genetic backgrounds. We have seen that extreme susceptibility is heritable and survives rederivation into SPF conditions, a procedure that would be expected to cause a change of microbial flora. Moreover, the phenotype that we observe is stable and continues to occur at a very high frequency in Il1rn−/− animals in our colony more than 2 yr since its first detection. It is unlikely that we have selected a susceptibility-causing mutation, unless it is present in the E14 TG2A stem cell line, because our second Il1rn-null line on an MF1 × 129/Ola background has also developed disease, yet the MF1 came from a separate colony and the chimera was derived from a different G418-resistant clone.
Because heterozygotes develop mild disease, we suggest that the IL-1ra gene is haploinsufficient. This is plausible because of the unusual function of the IL-1ra protein, acting solely as a competitive inhibitor of IL-1. Its concentration at an inflamed site is therefore likely to be critical to its efficacy, and, in the absence of sufficient compensatory regulation, it is likely to achieve a maximum in the heterozygote of only half of the wild-type level. An alternative explanation, however, might be that we have unwittingly created a hypomorphic allele, although there is no obvious reason for the truncated open reading frame of IL-1ra (which is a single domain protein) to be expected to yield a dominant negative species.
We conclude that IL-1ra is required in our mouse colony to prevent the development of lethal arteritis. Because the only known function of IL-1ra is to antagonize the activity of IL-1, our working hypothesis is that the disease is caused by the unopposed action of IL-1. IL-1 induces a wave of release, from many cells within the affected tissue, of chemokines and proinflammatory cytokines that would recruit further inflammatory cells. In the absence of IL-1ra activity, this would amplify the unbalanced response. IL-1 might initially be released spontaneously by stressed smooth muscle or endothelial cells or by macrophages (possibly initially triggered by bacterial infection or oxidized lipid deposition) in the walls of high pressure vessels in areas of high turbulence. We have shown that macrophages are abundant in established lesions, many having cytoplasmic loads of IL-1, and that CD4+
T cells are present. It seems very likely that later IL-1 production from macrophages in established lesions occurs as a result of their activation by CD4+
T cells, and we speculate that the latter might be autoreactive. We have found no evidence of an autoreactive humoral response. We are currently confirming the essential role of IL-1 in the phenotype by crossing Il1rn
-null animals with mice that lack the IL-1 receptor 35
. The disease we observe in IL-1ra–deficient mice may offer an opportunity to dissect important pathophysiological mechanisms occurring in large arteries.