Scand. J. Immunol. 35, 361-367. 1992
Murine Intestinal Humoral Responses in Chronic Schistosoma mansoni Infections J. E. C R A B T R E E . C. E. PULLAR*. L. K. TREJDOSIEWfCZ & R. A. WILSON* Department of Medicine, St James's University Hospital, Leeds and *Departmcnt of Biology. University of York, York, UK Crabtree JE, Pullar CE.Trejdosiewicz LK. Wilson RA. Murine Intestinal Humoral Responses in Chronic Sehistosoma mansoni Infections. Scand J Immunol 1992:35:361-7 Specific and non-specific production of immunoglobulins (Ig) by the intestinal mucosa was examined in mice infected with the human blood fluke Schi.stosoma mansoni. Heal and colonic mucosal tissue samples were cultured for 2 days, the medium replaced and the culture continued for a further 2 days. Ig concentrations and specific antibodies to soluble schistosome egg antigens in culture supernatants were estimated by isotype-specitic ELISA. Cultured mucosae from control mice produced little IgG. but signiticant amounts of IgA and IgM or prolonged culture. IgG concentrations were increased in infected animals, mainly in the initial culture period, indicative of systemic, ralher than local origins. By contrast, signiticantly increased local production of IgA and IgM occurred alter ihe start of egg deposition in the intestinal mucosae. Although specific anti-egg antibodies ofthe IgG and IgM class were detccled, none of the local IgA response was specific for schistosome eggs. We conclude thai specific intestinal immune responses lo schistosome eggs reflect systemic responses, whereas locally increased IgA production is largely non-specific. This pattern of response is likely to be related to the prior systemic exposure lo schistosome eggs, which results in polyclonal local B-cell activalion. but fails to trigger an antigen-specific IgA mucosal response. Dr J. E- Crabtree. CSB Level 7, Departmeni of Medicine. St James'.? University Hospital. Leeds. LS9 7TF. UK
Chronic Schisto.wma mansoni infcclions in both animal models and man induce a strong systemic immune response against the antigens of schistosomula [I], adult worms [2. 3] and eggs [4]. In addition, as in many other parasitic infections, polyclonal stimulation of humoral responses occurs [4]. As with other helminth infections, considerable antigen exposure occurs at mucosal sites. Exposure occurs in the lungs during schistosomular migratory phases [5.6] and the intestines, a site of egg deposition.
responses may be important in the development of IgA nephropathy [11]. In this study, we have exatnined the changes in intestinal humoral responses during the development of chronic S. mansoni infections in mice. Heal and colonic biopsies have been cultured in vitro and the secretion of total IgG, IgM and IgA and class-specific anli-soluble egg antigen antibodies determined by ELISA.
Although faecal egg output in experimental S. /fi(jH.v(W( infections reaches a peak at 8 weeks postinfection, oviposition is thought to be relatively constant [7]. The intestinal humoral responses to ,S'. mansoni egg deposition have not been examined, although there is evidence that the granulomatous responses to eggs differ in the small and large intestine [8, 9]. The intestine is a major source of IgA production [10] and intestinal IgA
MATERIALS AND
METHODS
Biological material. A Puerto Rican strain ofScliisln.\onia nian.\oni was routinely maintained in Biomphalaria glahraia. Female Lac A mice, weighing 20 30 g. were infected percutaneously via the abdomen with 30 cercariae. In ritro eulture. Infected mice were killed by cervical dislocationat4.6.K. 10. 13and 15 weeks post-infection. Four mice were used at each time-point: six age- and
361
362
J. E. Crablree et at.
sex-matched mice were used as eoiilrols. Using aseptie Icchniques, intestinal hiopsies were lakcn from the terminal ileum and proxinml colon. Tissues were rinsed in slerile phosphate buffered saline (PB.S). After removal ofsurface fluid, biopsies were weighed: the mean wet weight was 0.22 g (median = 0.224. range = 0.087 0..196 g). Biopsies were cullnred in duplicate in 2 ml RPMI 1640 medium supplemented with 10"'., heat-inactivated letal ealf scrum and IOO /ig.l penicillin/streptomycin (Flow Laboratories. High Wycombe. Bucks.. UK) at 37 Cin a 5% CO2-humidi[ied atmosphere as previously described [12 15]. Supernatants were aspirated after 2 days and replaced with 2 ml fresh culture tnedium. Biopsies were cultured for a furlher 2 days. Culture supernatants were clarified by eentrifugation and stored at - 7 0 C until assay. 10//I Trasylol (Bayer. Newbury. Berks.. UK) were added to each sample to inhibit protease activity. The mesenterie lymph nodes were excised at ihe same time as the intesline and the weights ofthe mesenterie lymph nodes were obtained. Enzyme-linked immunosorhciU assays. 0) lotid immunoglohulins. The lolal IgG. IgM and IgA content of the medium afterculture wa.s determined by ELISA, as described previously [If*]. Flal-bottomed microtilre plates (Nunclon: Gibco. Paisley. UK) were coated with anti-mouse polyvalent immunoglobulins (Sigma Chemicals. Poolc, Dorse!. UK) at a concentration of 400 ng per well in O.I M bicarbonate buffer. pH 9.6. for l!< h at 4 C. Before use. piatcs were washed ihree times in washing bulfer |PBS containing 0.115"'" Tween 20). Appropriate dilulions of culture supernatants and a standard mouse reference serum (ICN Immunobiologicais. High Wyeombe. Bucks.. UK| were made in PBS eontaining 10'^. goat serum; supernalants and the reference standard were incubated in 100-/(1 volumes in lripiieatefor2 h. After washing as above, 100/d ofa pretilred dilution of peroxidase-coiiiugated goal antimouse IgG (IIOOOI. IgM (I 1000) or IgA (i.'lOO) in PBS,iO'*Ai goat serum was added to each well for 2 h. Afler washing as above, peroxidase activity was dctecled using orthophenylenediamine subsirale {Sigma)at 1 mg/ml (lOO/d per well). The reaction was stopped wilh X M H2SO4 (30 /i! per well) and the absorbance quantified on a plate reader (Dynatech, Billingshurst. Sussex. UK) at 490 nm. The amounts of secreted Ig were estimated from standard curves of Ihe mouse reference serum and Ihe resuils expressed in "/ig Ig.lOO mg biopsy (we! weight)'. Assay sensitivity for IgG. IgM and IgA was K.74. 13.4 and 41.4 ng/ml. respectively. (ii) Soluble c^g antigen specific aiuihudics. AntiScliistosoma mansoni egg antibodies in culture supernatanis were assayed by F.l.iSA. Soluble egg antigens (SEA) were prepared as previously de.seribed [17] by mechanical homogenisalion of isolaled eggs, followed by low speed cenlrifugation. Supernatants were recenlrifuged at 20.000 g (4 h at 4 C) and 0.45 ,im membrane filtered prior to determination of protein concentration. Flat-bottomed mierotitrc plales were coaled with 0.9 tig per well of SFA diluted in 0.1 M bicarbonate bulTer. as above. Optimum coating antigen eoncentrations were deicrmined by chequer-board tilrations against sera from chronically infected miee. Culture supernatanis were assayed by FLISA lor the
Wet weight (g) 0.6
I JIJJ 0
1
2
3
4
5
6
7
8
9
1 0 1 1 1 2 13 1 4 1 S
Weeks of Infection
FIG. I. Wel weight (mean + SEM) of mesenterie lymph nodes followmg .V. mansoni infection: control »( = 6, experimental n = 4. Values were signilicantly different from controls (/•
Murine Intestinal Humoral Responses in Chronic Schistosoma mansoni Infections J. E. C R A B T R E E . C. E. PULLAR*. L. K. TREJDOSIEWfCZ & R. A. WILSON* Department of Medicine, St James's University Hospital, Leeds and *Departmcnt of Biology. University of York, York, UK Crabtree JE, Pullar CE.Trejdosiewicz LK. Wilson RA. Murine Intestinal Humoral Responses in Chronic Sehistosoma mansoni Infections. Scand J Immunol 1992:35:361-7 Specific and non-specific production of immunoglobulins (Ig) by the intestinal mucosa was examined in mice infected with the human blood fluke Schi.stosoma mansoni. Heal and colonic mucosal tissue samples were cultured for 2 days, the medium replaced and the culture continued for a further 2 days. Ig concentrations and specific antibodies to soluble schistosome egg antigens in culture supernatants were estimated by isotype-specitic ELISA. Cultured mucosae from control mice produced little IgG. but signiticant amounts of IgA and IgM or prolonged culture. IgG concentrations were increased in infected animals, mainly in the initial culture period, indicative of systemic, ralher than local origins. By contrast, signiticantly increased local production of IgA and IgM occurred alter ihe start of egg deposition in the intestinal mucosae. Although specific anti-egg antibodies ofthe IgG and IgM class were detccled, none of the local IgA response was specific for schistosome eggs. We conclude thai specific intestinal immune responses lo schistosome eggs reflect systemic responses, whereas locally increased IgA production is largely non-specific. This pattern of response is likely to be related to the prior systemic exposure lo schistosome eggs, which results in polyclonal local B-cell activalion. but fails to trigger an antigen-specific IgA mucosal response. Dr J. E- Crabtree. CSB Level 7, Departmeni of Medicine. St James'.? University Hospital. Leeds. LS9 7TF. UK
Chronic Schisto.wma mansoni infcclions in both animal models and man induce a strong systemic immune response against the antigens of schistosomula [I], adult worms [2. 3] and eggs [4]. In addition, as in many other parasitic infections, polyclonal stimulation of humoral responses occurs [4]. As with other helminth infections, considerable antigen exposure occurs at mucosal sites. Exposure occurs in the lungs during schistosomular migratory phases [5.6] and the intestines, a site of egg deposition.
responses may be important in the development of IgA nephropathy [11]. In this study, we have exatnined the changes in intestinal humoral responses during the development of chronic S. mansoni infections in mice. Heal and colonic biopsies have been cultured in vitro and the secretion of total IgG, IgM and IgA and class-specific anli-soluble egg antigen antibodies determined by ELISA.
Although faecal egg output in experimental S. /fi(jH.v(W( infections reaches a peak at 8 weeks postinfection, oviposition is thought to be relatively constant [7]. The intestinal humoral responses to ,S'. mansoni egg deposition have not been examined, although there is evidence that the granulomatous responses to eggs differ in the small and large intestine [8, 9]. The intestine is a major source of IgA production [10] and intestinal IgA
MATERIALS AND
METHODS
Biological material. A Puerto Rican strain ofScliisln.\onia nian.\oni was routinely maintained in Biomphalaria glahraia. Female Lac A mice, weighing 20 30 g. were infected percutaneously via the abdomen with 30 cercariae. In ritro eulture. Infected mice were killed by cervical dislocationat4.6.K. 10. 13and 15 weeks post-infection. Four mice were used at each time-point: six age- and
361
362
J. E. Crablree et at.
sex-matched mice were used as eoiilrols. Using aseptie Icchniques, intestinal hiopsies were lakcn from the terminal ileum and proxinml colon. Tissues were rinsed in slerile phosphate buffered saline (PB.S). After removal ofsurface fluid, biopsies were weighed: the mean wet weight was 0.22 g (median = 0.224. range = 0.087 0..196 g). Biopsies were cullnred in duplicate in 2 ml RPMI 1640 medium supplemented with 10"'., heat-inactivated letal ealf scrum and IOO /ig.l penicillin/streptomycin (Flow Laboratories. High Wycombe. Bucks.. UK) at 37 Cin a 5% CO2-humidi[ied atmosphere as previously described [12 15]. Supernatants were aspirated after 2 days and replaced with 2 ml fresh culture tnedium. Biopsies were cultured for a furlher 2 days. Culture supernatants were clarified by eentrifugation and stored at - 7 0 C until assay. 10//I Trasylol (Bayer. Newbury. Berks.. UK) were added to each sample to inhibit protease activity. The mesenterie lymph nodes were excised at ihe same time as the intesline and the weights ofthe mesenterie lymph nodes were obtained. Enzyme-linked immunosorhciU assays. 0) lotid immunoglohulins. The lolal IgG. IgM and IgA content of the medium afterculture wa.s determined by ELISA, as described previously [If*]. Flal-bottomed microtilre plates (Nunclon: Gibco. Paisley. UK) were coated with anti-mouse polyvalent immunoglobulins (Sigma Chemicals. Poolc, Dorse!. UK) at a concentration of 400 ng per well in O.I M bicarbonate buffer. pH 9.6. for l!< h at 4 C. Before use. piatcs were washed ihree times in washing bulfer |PBS containing 0.115"'" Tween 20). Appropriate dilulions of culture supernatants and a standard mouse reference serum (ICN Immunobiologicais. High Wyeombe. Bucks.. UK| were made in PBS eontaining 10'^. goat serum; supernalants and the reference standard were incubated in 100-/(1 volumes in lripiieatefor2 h. After washing as above, 100/d ofa pretilred dilution of peroxidase-coiiiugated goal antimouse IgG (IIOOOI. IgM (I 1000) or IgA (i.'lOO) in PBS,iO'*Ai goat serum was added to each well for 2 h. Afler washing as above, peroxidase activity was dctecled using orthophenylenediamine subsirale {Sigma)at 1 mg/ml (lOO/d per well). The reaction was stopped wilh X M H2SO4 (30 /i! per well) and the absorbance quantified on a plate reader (Dynatech, Billingshurst. Sussex. UK) at 490 nm. The amounts of secreted Ig were estimated from standard curves of Ihe mouse reference serum and Ihe resuils expressed in "/ig Ig.lOO mg biopsy (we! weight)'. Assay sensitivity for IgG. IgM and IgA was K.74. 13.4 and 41.4 ng/ml. respectively. (ii) Soluble c^g antigen specific aiuihudics. AntiScliistosoma mansoni egg antibodies in culture supernatanis were assayed by F.l.iSA. Soluble egg antigens (SEA) were prepared as previously de.seribed [17] by mechanical homogenisalion of isolaled eggs, followed by low speed cenlrifugation. Supernatants were recenlrifuged at 20.000 g (4 h at 4 C) and 0.45 ,im membrane filtered prior to determination of protein concentration. Flat-bottomed mierotitrc plales were coaled with 0.9 tig per well of SFA diluted in 0.1 M bicarbonate bulTer. as above. Optimum coating antigen eoncentrations were deicrmined by chequer-board tilrations against sera from chronically infected miee. Culture supernatanis were assayed by FLISA lor the
Wet weight (g) 0.6
I JIJJ 0
1
2
3
4
5
6
7
8
9
1 0 1 1 1 2 13 1 4 1 S
Weeks of Infection
FIG. I. Wel weight (mean + SEM) of mesenterie lymph nodes followmg .V. mansoni infection: control »( = 6, experimental n = 4. Values were signilicantly different from controls (/•
