K.S. Ovington

IL-5-Deficient Mice Have a Developmental Defect in CD5+ B-1 Cells and Lack Eosinophilia but Have Normal Antibody and Cytotoxic T Cell Responses

Mice deficient in interleukin-5 (IL-5-/- mice) were generated by gene targeting in embryonal stem cells. Contrary to previous studies, no obligatory role for IL-5 was demonstrated in the regulation of conventional B (B-2) cells, in normal T cell-dependent antibody responses or in cytotoxic T cell development. However, CD5+ B cells (B-1 cells) in the peritoneal cavity were reduced by 50%-80% in 2-week-old IL-5-/- mice, returning to normal by 6-8 weeks of age. The IL-5-/- mice did not develop blood and tissue eosinophilia when infected with the helminth Mesocestoides corti, but basal levels of eosinophils with normal morphology were produced in the absence of IL-5. IL-5 deficiency did not affect the worm burden of infected mice, indicating that increased eosinophils do not play a significant role in the host defence in this parasite model.

The role of eosinophils in parasitic helminth infections: insights from genetically modified mice.

Eosinophilia - an increase in the number of eosinophils in the blood or tissues - has historically been recognized as a distinctive feature of helminth infections in mammals. Yet the precise functions of these cells are still poorly understood. Many scientists consider that their primary function is protection against parasites, although there is little unequivocal in vivo evidence to prove this. Eosinophils are also responsible for considerable pathology in mammals because they are inevitably present in large numbers in inflammatory lesions associated with helminth infections or allergic conditions. In this review, Carolyn Behm and Karen Ovington outline some of the cellular and biological properties of eosinophils and evaluate the evidence for their role(s) in parasitic infections.

Cytokines and immunological control of Eimeria spp.

Protozoan parasites belonging to the genus Eimeria cause considerable losses in livestock production in which stocking densities are high or environments restricted. The ability of hosts to mount immunological responses which limit parasite reproduction vary according to the particular species of Eimeria. Typically though, immune responses restrict parasite reproduction during primary infection and limit, if not prevent, subsequent infections. Although mechanisms of immunity are unknown, host immune responses have been exploited in the development of a method to control coccidiosis-immunisation with attenuated strains of Eimeria. Limitations of this control method, predominantly the cost of producing the attenuated parasites, necessitates identification of protective immune responses to facilitate selection of antigens for use in non-living vaccines. As in immune responses to many other parasitic infections of the gastrointestinal tract, the role of antibodies is at best minor, whereas T-cells are crucial. Numerous studies have shown that the intestinal mucosal T-cell population is dynamic; the number and phenotype of T-cells changes in response to Eimeria-infection. Specific changes in the intestinal T-cell population have not, however, been correlated with limitation of parasite reproduction. Experiments involving adoptive transfer of T-cell sub-populations and in vivo depletion of specific T-cells have shown that CD4+ T-cells and to a lesser extent CD8+ T-cells are important in immune responses which limit primary infection. In contrast, CD8+ T-cells are more important in subsequent infections with CD4+ T-cells having a lesser role. The effects of T-cells on Eimeria are partially mediated by the cytokines they release. Most attention has concentrated on interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) because these cytokines have been shown to limit other protozoan infections. IFN-gamma is produced in Eimeria-infected hosts but evidence that it is present at the site of infection is limited. Intestinal levels of IFN-gamma increase earlier in response to primary Eimeria-infection in mice which are relatively resistant, than in mice which are relatively susceptible. Neutralisation of endogenously produced IFN-gamma has shown that this cytokine limits oocyst production in either primary or secondary infections depending on the species of Eimeria. Production of TNF-alpha is also increased in infected hosts. In comparison with relatively susceptible mice, TNF-alpha is produced earlier and to a greater extent in the intestines of relatively resistant mice. Unexpectedly, injections of TNF-alpha into infected mice increased oocyst production. It remains to be determined whether the effects of endogenous TNF-alpha are the same as those of exogenous TNF-alpha. Mechanisms by which IFN-gamma and TNF-alpha modulate parasite reproduction have not been identified. A number of lines of experimentation have suggested that it is unlikely that IFN-gamma limits parasite reproduction through induction of the synthesis of reactive oxygen or reactive nitrogen intermediates, since both of these reactive intermediates have the capacity to exacerbate Eimeria-infection.

The enigmatic eosinophil: investigation of the biological role of eosinophils in parasitic helmint infection

In many helminth infected hosts the number of eosinophils increases dramatically, often without any concurrent increases in the number of other leukocytes, so that eosinophils become the dominant cell type. Many experimental investigations have shown that the eosinophilia is induced by interleukin-5 (IL-5) but its functional significance remains unclear. Mice genetically deficient in IL-5 (IL-5-/-) have been used to evaluate the functional consequences of the IL-5 dependent eosinophilia in helminth infected hosts. Host pathology and level of infection were determined in IL-5-/- and wild type mice infected with a range of species representative of each major group of helminths. The effects of IL-5 deficiency were very heterogeneous. Of the six species of helminth examined, IL-5 dependent immune responses had no detectable effect in infections with three species, namely the cestodes Mesocestoides corti and Hymenolepis diminuta and the trematode Fasciola hepatica. In contrast, IL-5 dependent immune responses were functionally important in mice infected with three species, notably all nematodes. Damage to the lungs caused by migrating larvae of Toxocara canis was reduced in IL-5-/- mice. Infections of the intestine by adult stages of either Strongyloides ratti or Heligmosomoides polygyrus were more severe in IL-5-/- mice. Adult intestinal nematodes were clearly deleteriously affected by IL- 5 dependent processes since in its presence there were fewer worms which had reduced fecundity and longevity. The implications of these results for the viability of using inhibitors of IL-5 as a therapy for asthma are considered.

Fasciola hepatica: Free radical generation by peritoneal leukocytes in challenged rodents

Free radical generation by peritoneal leukocytes from hosts able to develop resistance to reinfection with Fasciola hepatica (rats) was compared with that of hosts unable to develop resistance (mice). Free radical generation by rat leukocytes was 3.5 times higher per cell and 30 times higher per animal than radical production by mouse leukocytes. The capacity of peritoneal leukocytes to produce free radicals in response to adult fluke crude antigen was increased by the presence of host plasma and was quantitatively greater in challenged rats than in naive or primary infected rats. This was not the case for mice, in which cells from primary infected animals were equally as responsive as cells from challenged mice. Further experiments revealed that challenge infection in rats apparently caused the in vivo activation of peritoneal leukocytes and increased levels of unidentified factors in plasma and that both of these responses were involved in the initiation of free radical generation in response to F. hepatica. Dramatic increases in the number of eosinophils present in the peritoneal cavities of primary infected and challenged rats (but not mice) were observed but the role of eosinophils in the production of free radicals in response to F. hepatica remains to be determined.

Cytokines, free radicals and resistance to Eimeria

The cytokine, gamma-interferon (IFN-gamma), which is produced by CD4(+) T cells, plays a crucial role in host resistance to Eimeria infections. Karen Ovington and Nick Smith propose that free oxygen radical generation by leukocytes in response to infection with Eimeria is the result of activation by IFN-gamma. The functional role of free oxygen radicals is unclear but these highly reactive radicals are produced by the leukocytes that infiltrate the intestine in large numbers during infection, and the parasites,enterocytes and cells of the immune system may all be vulnerable to oxidative damage. Gamma-interferon also appears to induce the enterocytes inhabited by Eimeria to turn against the parasite. The authors draw from literature documenting similar effects on other protozoa, especially Leishmania and Plasmodium, and speculate that reactive nitrogen intermediates produced by enterocytes have a functional role in resistance to Eimeria.

The role of carbon dioxide in the formation of end-products by Hymenolepis diminuta.

Abstract The hypothesis that ambient CO 2 levels determine the end-products of energy metabolism excreted by Hymenolepis diminuta was tested by incubating the parasite in a range of CO 2 concentrations and measuring internal concentrations of adenine nucleotides and the excretion of organic acids. The strain of H. diminuta used was found to excrete mainly lactic acid and acetic acid. Succinic acid production was generally less than 5–10% of the total. At high CO 2 concentrations, the rate of excretion of lactic acid decreased while that of succinic acid increased, which conforms with the hypothesis. Acetic acid excretion did not vary significantly over the range of CO 2 concentrations used. Other results did not support the hypothesis. High CO 2 levels reduced the total amounts of acids excreted and the rate of succinic acid excretion was so small as to be ineffective in preventing the accumulation of H + ions. When present in the incubation medium, succinic acid was taken up by H. diminuta . Lactic and acetic acid excretion was always sufficient to limit the accumulation of H + ions. The conditions of incubation were shown not to be responsible for the low rates of succinic acid excreted. Incubation conditions and metabolic end-products were found to affect the rates of excretion of organic acids. There is thus a need, in work of this nature, to regulate and specify experimental conditions and to stipulate the strain of parasite used. The hypothesis was rejected and it was suggested that the energy metabolism of parasitic helminths is adapted to fluctuating O 2 and CO 2 tensions.

Free radical generation and the course of primary infection with Nippostvongylus brasiliensis in congenitally athymic (nude) rats

Summary The course of primary infections with Nippastrongylus brasiliensis was followed in nude (CBH/R nu/nu) and heterozygote(CBH/R nu/+) rats. In both nude and heterozygote rats peak egg production by N. brasiliensis occurred on days 7 and 8 post‐infection. However, whereas in heterozygote rats egg production declined rapidly thereafter and ceased completely by day 14 post‐infection, in nude rats high numbers of N. brasiliensis eggs were still seen on day 27 post‐infection, when the experiment was terminated, in comparison with the nude rats, heterozygote rats had a 5‐fold greater loss of weight by day 9 post‐infection and a 4‐fold higher incidence of diarrhoea. Furthermore, infected heterozygote rats became anaemic whereas uninfected rats and infected nude rats showed no evidence of anaemia. Free radical generation was measured in infected (9 days) and uninfected rats. Leucocytes from infected heterozygote rats were able to generate copious quantities of free radicals in response to N. brasiliensis whereas leucocytes from infected nude rats produced only slightly more free radicals than uninfected rats. Thus, worm rejection, weight loss, diarrhoea, anaemia and free radical generation in response to N. brasiliensis infection arc all T‐cell dependent events.

Cytokine and immunoglobulin subclass responses of rats to infection with Eimeria nieschulzi

SIV rats infected with a high dose (50,000 oocysts) of Eimeria nieschulzi displayed clinical symptoms of coccidiosis such as diarrhoea (days 6 and 7 post-primary infection) and weight loss (days 6-8 post-primary infection) and were completely immune to challenge with a similar dose. The ability of rats to produce tumour necrosis factor (TNF) in vivo was enhanced during the period of oocyst excretion in the primary infection but significant production of TNF did not occur after challenge infection. Thus, TNF does not appear to be an important factor in resistance to infection with E. nieschulzi but may play some role in resistance to primary infection and in the pathology associated with E. nieschulzi infection. Parasite-specific serum IgM levels (measured by enzyme-linked immunosorbent assay) were also increased during primary infection but returned to background levels at the end of the patent period and were not affected by challenge infection. In contrast to TNF and IgM, serum concentrations of E. nieschulzi-specific IgG1, IgG2a, IgG2b, IgG2c and intestinal tissue levels of IgA did not begin to increase until after day 12 post-primary infection, reached peak levels between days 20 and 30 post-primary infection and were slightly increased by challenge infection.

THE EFFECT OF BCG, ZYMOSAN AND COXIELLA BURNETTI EXTRACT ON EIMERIA INFECTIONS

Infection of animals with species of Eimeria induces a hyper‐reactivity to endotoxin as manifest by a greatly increased capacity of infected animals to produce TNF in response to LPS in vivo compared with uninfected animals. This finding indicates priming for hyperactivation of macrophages by Eimeria infection and raises the possibility that non‐specific triggering of macrophages by agents such as Bacille Calmette‐Guerin (BCG), zymosan or Coxiella burnetti extract may be a simple means of control for coccidiosis. However, all of these agents enhanced oocyst excretion in mice, rats or chickens infected with Eimeria vermiformis. Eimeria nieschulzi or Eimeria tenella, respectively, without affecting the patent period.