Scand. J. Immutwl. 9, 23-28. 1979
Selective Expression of Antibody Classes and Contact Sensitivity Affected by Genes in the Major Histocompatibility Complex W. R. THOMAS. M. C. WATKINS & G. L. ASHERSON Division of Immunological Medicine, Clinical Research Centre, London, UK
Thomas, W.R., Watkins, M.C. & Asherson, G.L. Selective Expression of Antibody Classes and Contact Sensitivity Affected by Genes in ihe Major Hisiocompatihilily Complex. Scand. J. Immunol. 9, 23-28, 1979. This report describes IgM. igG and IgF. antibody and contact sensitivity responses of strains of mice congenic at the major hislocompatibility complex (MHC) to skin painting with picryl ehloride or oxuzolone. BIO had low responses of all classes to picryl chloride. This was also retlectet! hy ihe DNA synthesis occurring in their draining lymph nodes after painting. BIOBR were high responders to picryl chloride for all classes but BIOA and BU)D2 «crc high re^ponders for all classes except igE. This presents evidence that genes in the MMC can selectively control antibody classes. The contact sensitivity response of the congenics to oxazolone confirmed the low previously described responsiveness of BIO mice. Antibody responses to oxa?olone (agglutinin and reagin) were low for all congenics wilh BIO backgrotiiids. If. R. Thomas, Divisi.m of Imniunohigiatl Medicine, WatftrJ Road. Harrow. Midille.K-.v IIAI 3UJ, UK.
Mice painted on the skin with the contactsensitizing agents picryl chloride or oxazolone produce IgM, IgG and IgE antibody [27] as well as contact sensitivity. Regulatory effects of the major hislocompatibility complex (MHC) and other loci on the contact sensitivity to picryl chloride [23] and oxazolone [7] and on the IgM and IgG antibody responses to oxazolone [2] have been described. Our investigations into cellular mechanisms involved in the responses to picryi chloride have shown that antibody and contact sensitivity can be selectively regulated by cells [26] and also that the IgG antibody response could be inhibited independently of IgE responses [28]. It was therefore of interest to study genetic effects on the expression of contact sensitivity and antibody classes. This report describes responses of MHC-congenic mice to picryl chloride and oxazolone and shows that genes in the MHC can selectively affect different classes of immunity. In particular, it shows that MHC-congenic mice could produce IgE titres while having the same IgG responses.
MATERIALS AND METHODS Mice. ii-l2-week-o!d niiiles from the hrccding unit of ihc Clinical Research Centre or from OLAC (1976) Ltd, Oxfordshire, were used. Aiitihi'ily. Agghitinin.s. Sera were absorbed with glutaraldehyiJe-lrealed sheep erythrocytes (SRBC) and titrated with a microtitre apparatus against glutaraldchyde-lreaied trinitrophenyl (TNP) or oxazolone (OXJcoupled SRBC. Titres iire e.\presscd as log.^ of the reciprocal of the last dilution showing agglutination. Treatment with 2-mcrcaptoethanol (Ml-) (BDH Chemicals Ltd, Poole) was used to distinguish IgG [27. 28]. Passive cutaneous anaphylaxis {PCA]. Mouse reagin titres were determined by PCA in rat skin using a 24 h sensitization period [27]. Sera were titrated as doubling dilutions and ihe titre taken as logo of the reciprocal of the last dilution producing PCA. In the experiments described here sera were prcdilutcd 1/8. and the results arc shown as titres from this dilution, i.e. a titre of 4 represents a positive PCA at 1/64. Contact sensitivity. Mice were challenged by painting both sides of lioth ears with either !"„ picryl chloride (HPH, Poole, Dorset) or 1"^ oxazotone (2-phenyl-4eihoxymclhylene oxazolonet (BDH) in olive oil, and the increase in ear thickness produced by this challenge was measured with an engineer's micrometer afier 24 h. Results are in units of 10 •' cm.
0300-9405/79/0100-0023 $02.00 © 1979 Blackwell Scientific Publications
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. R. Thomas, M. C. Watkitts & G. L. A.slierson
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Assesstilcnt of DNA synthesis IIUDR incorporation). Mice were injected i.p. with 10"' M 5-fluoro-2-deo\yuridlne in 0.2 ml saline followed afier 30 min by 2 [iCi 5-iodo-['-'^l]2-deoxyuridine (lUDR). Lymph nodes and spleens were harvested after 2 h and washed three limes in 7O'\, ethanol over 3 days. lUDR incorporation is recorded as percentage injected radioactivity [4]. E.xpcrimentid procedure. Mice were painted on the abdomen, thora.x and forepaws with a total of O.I nil 5",, picryl chloride and afier 1 week challenged on the ears to be measured for contact sensitivity. The swelling was compared to that of an unsensitized group. Two weeks after the first painting the mice were given a second painting (same dose) and bled after 6 days lo measure antl-TNP antihody. Responses to oxazolone were determined by a similar regime, but the mice used for sensitization were the negalive controls of the picryl chloride experiment, and negative controls for oxazolone were the group sensitized to picryl chloride. The experiments were limci! so that 3 weeks lapsed between ear challenges.
RESULTS Responses of MHC congenics Responses of MHC congenic mice to picryl chloride and oxazolone are shown. AQR mice included in Ihis scries have not been back-crossed to ihe same extent as the other congenics [22].
Contact setisitivity (Table I) Responses to picryl chloride could be divided into small (BiO). moderate (B10D2, BIOBR. BiOA) and large (AQR). Differences in their responses to oxazolone could not be detected by the experimental procedure described above (data not shown), but because a previous report showed thai H-Z^' (BIO) congenics were lower responders than other strains [7], responses of BIO and BIOA to oxazolone were tested without prior exposure to picryl chloride. BIOA mice had an average specific swelling of 15.7 and BIO of 7.8 units (significance of difference P
Selective Expression of Antibody Classes and Contact Sensitivity Affected by Genes in the Major Histocompatibility Complex W. R. THOMAS. M. C. WATKINS & G. L. ASHERSON Division of Immunological Medicine, Clinical Research Centre, London, UK
Thomas, W.R., Watkins, M.C. & Asherson, G.L. Selective Expression of Antibody Classes and Contact Sensitivity Affected by Genes in ihe Major Hisiocompatihilily Complex. Scand. J. Immunol. 9, 23-28, 1979. This report describes IgM. igG and IgF. antibody and contact sensitivity responses of strains of mice congenic at the major hislocompatibility complex (MHC) to skin painting with picryl ehloride or oxuzolone. BIO had low responses of all classes to picryl chloride. This was also retlectet! hy ihe DNA synthesis occurring in their draining lymph nodes after painting. BIOBR were high responders to picryl chloride for all classes but BIOA and BU)D2 «crc high re^ponders for all classes except igE. This presents evidence that genes in the MMC can selectively control antibody classes. The contact sensitivity response of the congenics to oxazolone confirmed the low previously described responsiveness of BIO mice. Antibody responses to oxa?olone (agglutinin and reagin) were low for all congenics wilh BIO backgrotiiids. If. R. Thomas, Divisi.m of Imniunohigiatl Medicine, WatftrJ Road. Harrow. Midille.K-.v IIAI 3UJ, UK.
Mice painted on the skin with the contactsensitizing agents picryl chloride or oxazolone produce IgM, IgG and IgE antibody [27] as well as contact sensitivity. Regulatory effects of the major hislocompatibility complex (MHC) and other loci on the contact sensitivity to picryl chloride [23] and oxazolone [7] and on the IgM and IgG antibody responses to oxazolone [2] have been described. Our investigations into cellular mechanisms involved in the responses to picryi chloride have shown that antibody and contact sensitivity can be selectively regulated by cells [26] and also that the IgG antibody response could be inhibited independently of IgE responses [28]. It was therefore of interest to study genetic effects on the expression of contact sensitivity and antibody classes. This report describes responses of MHC-congenic mice to picryl chloride and oxazolone and shows that genes in the MHC can selectively affect different classes of immunity. In particular, it shows that MHC-congenic mice could produce IgE titres while having the same IgG responses.
MATERIALS AND METHODS Mice. ii-l2-week-o!d niiiles from the hrccding unit of ihc Clinical Research Centre or from OLAC (1976) Ltd, Oxfordshire, were used. Aiitihi'ily. Agghitinin.s. Sera were absorbed with glutaraldehyiJe-lrealed sheep erythrocytes (SRBC) and titrated with a microtitre apparatus against glutaraldchyde-lreaied trinitrophenyl (TNP) or oxazolone (OXJcoupled SRBC. Titres iire e.\presscd as log.^ of the reciprocal of the last dilution showing agglutination. Treatment with 2-mcrcaptoethanol (Ml-) (BDH Chemicals Ltd, Poole) was used to distinguish IgG [27. 28]. Passive cutaneous anaphylaxis {PCA]. Mouse reagin titres were determined by PCA in rat skin using a 24 h sensitization period [27]. Sera were titrated as doubling dilutions and ihe titre taken as logo of the reciprocal of the last dilution producing PCA. In the experiments described here sera were prcdilutcd 1/8. and the results arc shown as titres from this dilution, i.e. a titre of 4 represents a positive PCA at 1/64. Contact sensitivity. Mice were challenged by painting both sides of lioth ears with either !"„ picryl chloride (HPH, Poole, Dorset) or 1"^ oxazotone (2-phenyl-4eihoxymclhylene oxazolonet (BDH) in olive oil, and the increase in ear thickness produced by this challenge was measured with an engineer's micrometer afier 24 h. Results are in units of 10 •' cm.
0300-9405/79/0100-0023 $02.00 © 1979 Blackwell Scientific Publications
23
. R. Thomas, M. C. Watkitts & G. L. A.slierson
24
Assesstilcnt of DNA synthesis IIUDR incorporation). Mice were injected i.p. with 10"' M 5-fluoro-2-deo\yuridlne in 0.2 ml saline followed afier 30 min by 2 [iCi 5-iodo-['-'^l]2-deoxyuridine (lUDR). Lymph nodes and spleens were harvested after 2 h and washed three limes in 7O'\, ethanol over 3 days. lUDR incorporation is recorded as percentage injected radioactivity [4]. E.xpcrimentid procedure. Mice were painted on the abdomen, thora.x and forepaws with a total of O.I nil 5",, picryl chloride and afier 1 week challenged on the ears to be measured for contact sensitivity. The swelling was compared to that of an unsensitized group. Two weeks after the first painting the mice were given a second painting (same dose) and bled after 6 days lo measure antl-TNP antihody. Responses to oxazolone were determined by a similar regime, but the mice used for sensitization were the negalive controls of the picryl chloride experiment, and negative controls for oxazolone were the group sensitized to picryl chloride. The experiments were limci! so that 3 weeks lapsed between ear challenges.
RESULTS Responses of MHC congenics Responses of MHC congenic mice to picryl chloride and oxazolone are shown. AQR mice included in Ihis scries have not been back-crossed to ihe same extent as the other congenics [22].
Contact setisitivity (Table I) Responses to picryl chloride could be divided into small (BiO). moderate (B10D2, BIOBR. BiOA) and large (AQR). Differences in their responses to oxazolone could not be detected by the experimental procedure described above (data not shown), but because a previous report showed thai H-Z^' (BIO) congenics were lower responders than other strains [7], responses of BIO and BIOA to oxazolone were tested without prior exposure to picryl chloride. BIOA mice had an average specific swelling of 15.7 and BIO of 7.8 units (significance of difference P
