Suppression of heterologous immunity by Nematospiroides dubius antigens in vitro.

The direct effect of the soluble antigens in the homogenate of adult Nematospiroides dubius (AH) on spleen cells from uninfected NIH mice was investigated using a Mishell-Dutton culture system. Parasite antigens were shown to reduce the plaque-forming cell (PFC) response to sheep red blood cells (SRBC) in a dose-dependent manner in vitro. A population of suppressor cells was demonstrated in the spleens of infected mice. Furthermore naive spleen cells cultured in the presence of AH gave rise to cells which depressed the PFC response of naive cells when subsequently cultured together in vitro. Treatment of these cell populations with anti-thy 1.2 plus complement did not impair suppressor activity, and it was concluded that cells expressing the T-cell phenotype were not involved.


INTRODUCTION THE trichostrongyle nematode
Nematospiroides dubius survives in the mouse small intestine for 8 months or more in a primary infection (Ehrenford, 1954;Keymer & Hioms, 1986), and exerts a depressive effect on the host's ability to respond to heterologous (Chowaniec, Wescott & Congdon, 1972;Shimp, Crandall & Crandall, 1975;Price & Turner, l986a, b) and homologous antigens (Pritchard & Behnke, 1985;Sitepu, Dobson & Brindley, 1985). In mice concurrently infected with N. dubius and T. spiralis, the survival time of the latter parasite is prolonged compared to controls (Behnke, Wakelin & Wilson, 1978). A primary infection with N. dubius is also known to severely depress the host's immune response to concurrently administered sheep erythrocytes (Shimp et al., 1975; Ali & , and it can be demonstrated that the degree of suppression is a function of the number of adult worms present in the small intestine (Ali & Behnke, 1983). Suppression has also been shown to be maximal 14 days after infection, corresponding to the presence of the adult stage in the lumen, rather than the L4 stage which resides in the gut wall (Ah & Behnke, 1983). It has been proposed that N. dubius facilitates its own survival in the gastrointestinal tract in the face of an immune response by the production of i~unomodulato~ factors (Behnke, Hannah & Pritchard, 1983;Behnke, 19873 which may also be implicated in suppression of heterologous immunity. Thus mice given a normally immunogenic infection with irradiated larvae at the same time as an infection with normal larvae do not become immune to challenge , and homologous immunity stimulated by an anthelmintic abbreviated infection is reduced on injection with soluble adult antigen (Pritchard & Behnke, 1985), as is the response to the heterologous antigen, sheep red cells (SRBC) (Pritchard, Ah & Behnke, 1984).
The present study was designed to ~0~~ and expand these in vivo observations by utilizing a h&shell-Dutton culture system, in which normal splenocytes are stimulated in vitro with SRBC, and their responsiveness assessed in a plaque (PFC) assay. This system is advantageous in that it has proven to be more manipulable than in vivo systems, and requires significantly less material to modulate immune responsiveness.
MATERIALS AND METHODS Animals. Female NIH mice, 6-8 weeks old, bred under conventional animal house conditions, were chosen for this study following the demonstration that this strain was responsive to sheep erythrocytes using an in vitro Mishell-Dutton culture system. Antigens. Nematode antigens were prepared as described previously (Pritchard, Williams, Behnke & Lee, 1983) and filter sterilized prior to use. Sheep erythrocytes were kindly supplied by Mr Walker. School of Agricuiture, Nottingh~ University, Sutton Bonington. Anti-Thy-l.2 was obtained from Serotec Ltd, Blackthorn, Bicester, England. The optimum dose for complement mediated cytotoxicity of 99% of cells was predetermined using NIH mouse thymocytes, and was subsequently used at a concentration of 1 /I 000. Low toxicity rabbit complement was obtained from Cederlane Laboratories Ltd. Hornby, Ontario, Canada, and used at a dilution of 1 in 12.
In vitro culture. An in vitro assay to analyse the immunoregulatory effect of N. dubius antigens was developed essentially as described by Mishell & Dutton (1966, 1967. Briefly, spleen cells from female NIH mice were resuspended in RPMI 1640 medium supplemented with 5% fetal calf serum and antibiotics (I 00 i.u. per ml penicillin and 100 pg per ml streptomyciff) to give a final concentration of I X IO' cells per ml. To each well of the tissue culture tray 0.5 ml of this suspension was added, together with 0.5 ml of the parasite antigen at the required concentration, or with 0.5 ml of the second cell population (see results). The cultures were then inoculated with 30 yl of a 1% suspension of sheep erythrocytes, and incubated at 37°C in a humidified CO? incubator for 4 days. After this time, the cells were harvested and assayed for the presence of direct (IgM) plaque forming ceils against sheep erythrocytes using Cunnin~h~l's method ~~unnin~h~.

The effect of infection with N. dubius on the immune response of spleen cells to SRBC in vitro
From the data in Fig. 1 it can be seen that the immune responsiveness of the cell suspensions Female NIH mice were infected with 250 L3 larvae of N. dub&s on day 0 of the experiment, and their diminished as the infection progressed through the spleens removed on days 6,9, 15 and 20 post infection. The responsiveness of single cell suspensions larval stages to adul~ood on day 9. Fu~he~ore, prepared from the spleens to SRBC was then assayed splenocytes from mice harbouring adult parasites in vitro. Unchallenged cultures were tested in parallel.
(days 9-21) when co-cultured with naive cells from uninfected animals reduced the latter cell population's responsiveness to sheep erythrocytes in vitro.
The effect of adding the soluble antigens from the homogenate of adult N. dubius on the in vitro jejune response of norma~sp~eno~ytes to SRBC Normal splenocytes were inoculated with SRBC in the presence of A? dubius AH and their ability to respond was assessed 4 days later in a direct PFC assay. In addition, splenocytes cultured in the presence of AH for 4 days were washed thoroughly to remove any extraneous antigen, transferred to naive cell cultures, and their ability to suppress the SREK response of the naive cell population was investigated. receiving only sheep erythrocytes. However, the degree of suppression varied between experiments for a defined quantity of adult antigen, possibly because of differences in the batches of fetal calf serum and This result was explored further to investigate effector cells in this system, and the data in Table 2 SRBC. The crude worm homogenate caused suppresconfirm that addition of adult antigen to splenocyte cultures reduced the number of PFC observed to sion in this system, without any noticeable toxic effect sheep er~hrocytes compared to controls. When on cells (as measured by viability), even when used at normal spleen cells were incubated with 10 pg of AH for 4 days and then added to naive cell cultures high concentrations.
together with SRBC, the PFC response was reduced. It appears that the greater the number of AH-treated cells added, the more the PFC were suppressed. Supematants taken from cells incubated with adult antigen did not reduce the ability of normal splenocytes to respond to SRBC.   (13) *Each well contained 5 X 10" spleen cells from uninfected female NIH mice. microgrammes of N. dubius adult antigen was then different times after N. dubius infection to a heteroadded to separate cultures, either at the same time (C) logous antigen was investigated in vitro. This first as the sheep erythrocytes, or 1 (D), 2 (E) or 3 (F) days experiment (Fig. 1) confirmed the observations that later. Four days after initiation of the experiment, the mice harbouring adult worms (days 9-21) were less cells were harvested and assessed for antibody by the responsive to SREK than animals with a larval infec-PFC assay.
tion (day 6) (Ali & . Moreover, it was The results in Fig. 2 demonstrate that the addition of adult antigen concurrently with SRBC suppressed the PFC response of the splenocytes to the latter antigen. Suppression was detected irrespective of whether AH was added on days 0, 1,2 or 3, although the degree of suppression decreased with the increasing interval between the introduction of SRBC and adult antigen to the cultures.

Efleci of incubating AH-rrea~ed cells with anti-Thy 1.2 plus complement
Spleen cells from normal NIH mice were incubated for 4 days either in medium alone or with 10 lug AH, after which time they were harvested and washed three times to remove any extraneous antigen. Half of the AH-treated splenocytes were then incubated with anti-Thy 1.2 plus complement to remove any T-cells which were present. The three cell populations were added to naive spleen cell cultures together with SRBC, and the PFC response was assessed. Figure 3 shows that spleen ceils treated with N dubius adult homogenate when added to normal splenocytes reduced the PFC response of the latter cell population to SRBC. However, prior incubation of antigen treated spleen cells with anti-Thy 1.2 plus complement did not restore the PFC response to control levels.

DISCUSSION
The work presented in this paper set out to determine the direct effect of soluble adult N. dubius antigen on spleen cell responses to SRBC, in an attempt to understand further the mechanism by which suppression of heterologous immunity operates, and to pave the way for purification of immuno-mod~ato~ material from the parasite. Therefore the responsiveness of spleen cells taken from mice at FIG. 2. The effect of adding sotuble antigens from the homogenate of adult N. dubius (AH) to spleen cells from uninfected mice at different time intervals after stimulation by SRBC in vitro. 5 X loo cells from naive mice were incubated with (A-immunized control) or without (Bunstimulated control) 30 ~1 of 1% SRRC. Splenocytes stimulated by SREK in vitro were also cultured in the presence of 50 pg of AH, the latter being added to culture wells on day 0 (C), day 1 (D), day 2 (E) or day 3 (F) after initjation of incubation. Ail the cultures were harvested on day 4 for analysis of PFC. that splenocytes from mice harbouring adult parasites reduced the response of naive spleen cells to sheep erythrocytes, indicating the presence of a suppressor cell population in these animals. Similarly, soluble adult worm antigens were shown to directly reduce the in vitro PFC response of naive splenocytes in a dose dependent manner (Table 1). This also appeared to be due to a suppressor cell population, since the reduction in plaques of normal cells was a function of the transferred AH-treated cells and not the supernatants (Table 2). Furthermore, a reduction in the PFC response of splenocytes was observed even when adult antigen was added 3 days after inoculation of SRBC into Mishell-Dutton cultures (Fig. 2), indicating the rapid generation and influence of the suppressor cell on the production of IgM antibody to sheep erythrocpes. Treating cells incubated with adult N. dubius antigen with anti-Thy 1.2 plus complement to remove all T-cells did not eliminate the in vitro generated suppressor cell population, therefore T-cells are unlikely to be responsible. This conclusion is supported by the work of Price & Turner (1986a) who investigated in viva responses to ovalbumin administered with adjuvant in N. dubius infected mice. Variable suppressor activity was identified in spleen cells taken from infected mice transferred to irradiated recipients, which was not mediated by T-cells. However, a role for T-cells in suppression of heterologous immunity has possibly been implicated by the restorative effect of 2'-deoxy~~osine treatment on the immune response of N. dubius infected mice (Pritchard et al., 1984), although macrophage mediated suppression of the proliferative response of thymocytes to concanavalin A in rats can be abolished by 2 '-deoxyguanosine, but this has not yet been shown in mice (Bril, van den Akker, Hussaarts-Odijk & Benner, 1985). However, suppression is not the property of adult N. dub& antigen alone, for day 6 larval antigen was also able to reduce the PFC response in culture to SRBC, as were homogenates prepared from Necator americanus. Other nematode antigens such as those obtained from Ascaris suum gave different results, actually increasing the PFC response in vitro (personal observations).
Suppressor cells have been implicated as mediators of non-specific suppression to heterologous antigens in other parasitic infections. For example the response of mice infected with T. brucei to SRBC is depressed (Murray, Urquhart, Murray & Jennings, 1973) and in murine malarial infections of P. yoelii and P. be&ei impairment of humoral responses to sheep erythrocytes was observed (Salaman, Wedderburn & Bruce-Chwatt, 1969;Greenwood, Playfair & Torigiani, 1971). Experiments have shown that an excess of normal resident macrophages can profoundly depress lymph&yte proliferation, and if the macrophages are activated, the effect is even more pronounced (Kaufman, Simon & Hahn, 1982). Ail stages of N. dubius can activate complement by the alternative pathway (Prowse, Ey & Jenkin, 1979) so cleaving the complement component C, into C,a and C,b fragments, the latter of which is a potent agent for activating macrophages. Additionally the surface of many nematodes is polyanionic (Himmelhoch & Zuckerman, 1983;Murrell & Graham, 1983), a factor which can contribute to the activation of macrophages, and activated macrophages are known to be associated with chronic placation, such as occurs during infection with N. dubius (see Allison, 1978).
The possibility that a non-T suppressor cell might be involved in the non-specific suppression observed during infection of mice with T. brucei was raised by to SRBC. The experiment comprised the following groups: A. 5 X 10h naive snlenocvtes + SRBC + 5 X 10" naive cells after incubation in medium for 4 days. [Naive splenocytes from uninfected mice were incubated in medium without AH for 4 days but were otherwise treated identically to cells exposed to AH prior to addition to these cultures.] B. 5 X 10' naive splenocytes + SRBC + 5 X 10h AH treated cells. [Naive splenocytes were incubated in the presence of 10 pg of AH for 4 days prior to addition to these cultures.] C. 5 X 10h naive splenocytes + SRBC + 2.5 X 10h AH treated cells. [Naive splenocytes were incubated in the presence of 10 ,ug of AH for 4 days prior to addition to these cultures.]. D. 5 X IO" naive splenocytes + SRBC + 2.5 X 10h AH treated cells after incubation with anti-Thy Murray et al. (1973), who found proliferation of mononuclear phagocytes in the lymphoid organs of infected mice. Corsini, Clayton, Askonas & Ogilvie (1977) found that macrophages qbtained from mice profoundly reduced the ability of normal mouse spleen cells to proliferate and secrete antibody when cultured with lipopolysaccharide (LPS). Eardley & Jayawardena (1977) also observed the generation of adherent cells, able to depress antibody responses of normal spleen cells, present in spleens of T. brucei infected mice. However, other workers (Jayawardena, A.N. 1977. Abstract in Proceedings of the Vth International Congress on Protozoology, p. 72) have suggested that thymus dependent lymphocytes are involved in the generation of non-specific adherent suppressor cells during the course of murine malarial infections, and interactions of T-cells and macrophages have been involved in other suppressive effects, for example of contact sensitivity (Asherson & Zembala, 1974). It has been postulated that the triggering of the release of suppressor factors by macrophages may be antigen specific, while the factors themselves are non-specific (Allinson, 1978).
The significance of the observation that parasitic hehninths cause non-specific immunodepression in the host is not clear, but one possibility is that the organisms facilitate their own survival through reducing host immunocompetence (Ogilvie & Wilson, 1976;Behnke, 1987). However, parasite induced immunosuppression does not always prevent the host from responding to parasite antigens. For example, in Trypanosorna brucei infections, there was no evidence that the parasite caused any significant reduction of antibody responses in acute infections (MacAskiIl, Holmes, Jennings & Urquhart, 1981). Furthermore, in the case of N. dubius, mice infected with irradiated larvae, which generate good immunity to challenge, have a reduced capacity to respond to SRBC (Ali & Behnke, 1984). Thus the interrelationship of specific and non-specific immunodepression during N. dubius infection is still debatable and their relative importance to parasite survival is controversial.
One possibility may be that the role of immunomodulatory factors secreted by N. dubius is principally to incapacitate the host's defences in the intestine in the microenvironment created by the parasite (Pritchard & Behnke, 1985), and that the systemic non-specific effects are a consequence of this activity (Behnke, 1987). Further analysis of this system should reveal the precise relationship between parasite-specific and non-specific immunodepression in N. dubius infected mice, and the survival value of such strategies to the parasite.