Suppression of reaging antibody formation. IV. Suppression of reaginic antibodies to penicillin in the mouse.

Reaginic antibodies to the benzylpenicilloyl determinant (BPO) and ovalbumin (OA) were induced readily in B6D2F1 mice by a single i.p. injection of either 1 or 10 mug of BPO4-OA suspended with 1 mg of Al(OH)3 in 0.5 ml of saline. Administration of conjugates consisting of the hapten coupled to the isologous, nonimmunogenic murine gamma-globulins (MgammaG), i.e., BPO9-MgammaG, BPO11-MgammaG, or BPO12-MgammaG, resulted in complete and specific suppression of the induction of the anti-BPO reaginic antibody response without affecting, however, the level of reaginic antibodies to OA. Further study of the effect of epitope density on the immunologic properties of BPOX-MgammaG revealed that a) the lightly haptenated conjugated, BPO1-MgammaG and BPO2.9-MgammaG, were not immunosuppressive, b) the conjugates, BPO4.3-MgammaG and BPO19-MgammaG, were partially tolerogenic, and c) the heavily haptenated conjugate, BPO31-MgammaG, was nontolerogenic. Moreover, most importantly, the ongoing anti-BPO response in sensitized mice was readily abrogated by either four daily or four weekly injections of BPO9-MgammaG. The immunosuppressive effect of BPO12-MgammaG conjugates was dose dependent, complete suppression being achieved with 200 mug of the tolerogen. The unresponsiveness to BPO of spleen cells from immunosuppressed donors was also maintained in adoptive cell transfer experiments in spite of the additional administration of the immunizing antigen under conditions expected to yield a secondary IgE response. Hence, it is suggested that, with special precautions to prevent unleashing an anaphylactic shock, treatment of penicillin-sensitive individuals with polyvalent conjugates of an appropriate number of BPO groups per human gamma-globulin molecule would constitute a rational immunotherapeutic procedure for the abrogation of the allergic response to BPO.     

Specific suppression of anti-hapten reaginic antibody titers with hapten-coated liposomes

Reaginic antibodies to DNP and ovalbumin (OA) were induced in B6D2F1 mice by a single i.p. injection of 1 microgram of DNP3-OA suspended with 1 mg of A1(OH)3 in 0.5 ml of saline. The anti-DNP reaginic antibody titers were markedly depressed by treatment of mice with DNP-coated liposomes. This treatment, however, did not affect the level of antibody formation to OA.     

Carboxymethyl cellulose, a nonimmunogenic hapten carrier with tolerogenic properties.

This communication reports on the tolerogenic properties of carboxymethyl cellulose (CMC) as a nonimmunogenic carrier for 2.4 dinitrophenyl (DNP) and the benzylpenicilloyl determinant (BPO). Either normal or primed mice, given an optimal dose of 250 micrograms per animal of DNP CMC, when challenged with an immunogenic form of the hapten as early as 30 min or as late as 21 days thereafter were completely and specifically unresponsive to it. Experimental evidence suggests that this unresponsiveness is not due to suppressor cells. Furthermore, DNP CMC induces tolerance in vivo but fails to do so in vitro under conditions at which other tolerogenic carbohydrate hapten conjugates such as DNP-dextran do. This together with comparative studies of tolerance induction kinetics by DNP CMC and DNP-dextran in vivo led us to conclude that molecular properties other than the epitope density must be attributed to CMC's tolerogenic potential. CMC may also be used as a tolerogenic carrier for BPO with respect to IgE antibody production. Thus, normal or primed mice injected with the BPO CMC conjugate were found specifically unresponsive to a challenge with an immunogenic form of penicillin.     

The use of haptenated immunoglobulins to induce B cell tolerance in vitro. The roles of hapten density and the Fc portion of the immunoglobulin carrier

The relationship between the Fc region of trinitrophenylated (TNP)-immunoglobulins (Ig), and their ability to induce tolerance was examined. It was found that adult B cells responding to a T-independent (TI) antigen were tolerized by TNP11 human gamma globulin (HGG), but not by TNP10F(ab')2 fragments of HGG. Increasing the hapten density on the F(ab')2 fragments overcame their inability to induce tolerance. Thus, a TNP17-F(ab')2 was an effective tolerogen. Murine myeloma proteins of different IgG subclasses were similarly tested. A TNP12-IgG2a and a TNP11-IgG1 induced tolerance, whereas two TNP11-12-IgG3 did not. However, a more heavily haptenated TNP18-IgG3 was tolerogenic. These results suggest that lightly haptenated immunoglobulins depend upon Fc receptor binding to induce tolerance in adult B cells. Non-Fc receptor-binding carriers are not tolerogenic unless they are more heavily haptenated. Finally, T cell and macrophage depletion experiments suggest that the tolerogens act directly on the B cells.     

Tolerance induction during ontogeny. III. Carrier recognition by the immature and adult immune system determines tolerogenicity of hapten-carrier conjugates

Hapten-specific B-cell tolerance may be induced by nonimmunogenic hapten derivatives of carboxylmethylcellulose or methylcellulose (MC) in adult, neonatal, or irradiated fetal liver reconstituted mice. Such tolerance was shown to occur independent of T cells, and a receptor blockade has been ruled out as a causative mechanism. Oxidation and subsequent reduction of the vicinal hydroxyl groups of both carriers significantly reduces their tolerogenic potential in adult mice, yet their hapten derivatives remain nonimmunogenic. Such chemical modification of the carrier does not affect the molecular weight and not only does not reduce the binding avidity but increases it for either free antibody- or antigen-binding cells. We have examined the ability of the immature immune system to functionally discriminate between the nominal and the chemically modified hapten conjugate. Like adult mice, the immunologically immature animals were invariably capable of distinguishing between the tolerogenic and the nontolerogenic carrier. Mice treated during ontogenic development with 2,4-dinitrophenyl (DNP)-MC were found to be hapten specifically tolerant when challenged at 4 weeks of age with the TI-2 antigen DNP-Ficoll (F) but not when challenged with the polyclonal activator lipopolysaccharide (LPS) or the TI-1 antigen DNP-Brucella. Moreover, neonatal mice treated for 8 weeks with 2,4,6-trinitrophenyl-ovalbumin (TNP-OVA) were hapten specifically tolerant when challenged with TNP-OVA or the TI-1 antigen TNP-LPS but responded to a challenge with the TI-2 antigen TNP-F. These data suggest that B-cell tolerance in adult as well as in immunologically immature mice is not only carrier dependent but, in addition, that the carrier selects the B subpopulation to be rendered unresponsive. The most popular version of the clonal abortion hypothesis puts no constraints upon the nature of the antigen as long as the B cell is ontogenically "predisposed" toward being rendered unresponsive upon contact with a ligand of sufficiently high binding avidity. Our data are at variance with this prediction.     

Novel trifunctional carrier molecule for the fluorescent labeling of haptens

We developed a novel trifunctional carrier molecule for the synthesis of hapten-fluorophore conjugates as reporter molecules in immunoassays. This carrier eliminates some of the disadvantages associated with currently used fluorophore-labeling procedures including high nonspecific binding. The backbone of the carrier consists of the 21 amino acid residues of the insulin A-chain molecule. This polypeptide provides a single site (terminal amino group) for covalent coupling of the hapten, three carboxyl groups for the attachment of fluorophores, and four sulfhydryl groups for derivatization with hydrophilic residues to compensate for the hydrophobic effect of the attached fluorophores. The sites for fluorophore attachment are 4, 17, and 21 amino acids away from the hapten attachment site. This spatial separation minimizes quenching of the fluorescence signal due to interaction of the fluorophores with each other and with the attached hapten. In this study, 2,4-dinitrophenol (DNP) was selected as model hapten, fluorescein as label, and S-sulfonate groups as hydrophilic residues. The properties of the DNP-insulin A-chain-fluorescein conjugate (DNP-Ins-Fl) were compared to those of a DNP derivative labeled with a single fluorescein moiety via a small lysine spacer (DNP-Lys-Fl). The DNP-Ins-Fl conjugate exhibited a 3-fold lower nonspecific adsorption to immobilized non-immune IgG contributing to an approximately 3-fold more efficient displacement from the binding sites of an immobilized monoclonal anti-DNP antibody by the antigen DNP-lysine. Furthermore, at equimolar concentrations the DNP-Ins-Fl generated a 2.6-fold higher fluorescent signal than DNP-Lys-Fl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isotype-specific resistance against tolerance induction in SJL mice

Age-related changes in antibody response of SJL mice were examined in terms of isotype expression after treatment with immunogen or with immunogen, preceded by the molecule in normally tolerogenic form. We report here that tolerance induction and resistance to down regulation are isotype specific. Tolerance can be induced in terms of all detectable isotypes at the age of 5 weeks. In older SJL mice, tolerance to the carrier is found in IgM antibody, whereas there is resistance against down regulation in terms of IgG2a and IgG2b isotypes, and sensitization in terms of IgG3, IgG1, and IgA antibody. Furthermore, the degree of down regulation is determinant dependent. This was observed when older SJL mice, pretreated with the carrier in a normally tolerogenic form, were immunized with haptenated carrier and tested for their response to hapten and carrier determinants. In this case, IgA antibody shows tolerance to the hapten and sensitization by carrier determinants.     

Adaptive differentiation of murine lymphocytes. III. T and B lymphocytes display reciprocal preference for one another to develop optimal interacting partner cell sets.

Responses to the synthetic terpolymer L-glutanmic acid, L-lysine, L-phenylalanine (GLphi) and hapten derivatives thereof are controlled by two complementing H-2 linked Ir genes in the mouse. F1 hybrids derived from two different nonresponder strains (one of which possesses the alpha and the other beta Ir-GLphi gene) are phenotypic responders to GLphi and 2,4-dinitrophenyl (DNP)-GLphi. Moreover, spleen cells from DNP-GLphi-primed F1 mice can adoptively transfer secondary anti-DNP antibody responses to irradiate been challenged with DNP-GLphi. When, however, GLphi-primed F1 helper T cells are transfered together with the DNP-specific F1 B cells that had been primed in separate mice altogether by DNP coupled to an unrelated protein carrier, such mixtures failed to develop adequate adoptive secondary anti-DNP responses to DNP-GLphi. This contrasted with the ability of the same GLphi-primed F1 T cells to provide helper activity for DNP-primed B cells from responder recombinant B10.A (5R) mice. More important, the apparent defect of GLphi-primed F1 T cells in providing help for DNP-primed F1 B cells (primed to a DNP-protein conjugate) could be readily overcome by using DNP-primed B cells from donor F1 mice primed with DNP-GLphi. As discussed herein, these results suggest that interacting T and B lymphocytes pair off into partner cell sets, any pair of which interact optimally when a "best fit" reciprocal self-recognition occurs between them.     

Further studies on amplification of cell-associated immunological memory by secondary antigenic stimulus.

Using the hapten-carrier system in which the dinitrophenyl group (DNP) served as a B cell reactive hapten and bovine serum albumin (BSA) or human gammaglobulin (HGG) as a T cell reactive carrier, changes in the hapten-specific memory (B cell-associated memory) and the carrier-specific memory (T cell-associated memory) after a secondary antigenic stimulus were analyzed in mice. Since an immunological adjuvant was indispensable in the induction of the primary increase in memory, antigen used for the primary antigenic stimulus was injected together with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) which has already been shown to exhibit a potent adjuvant effect. With the cell-transfer technique, it was found that the cell-associated hapten-specific memory for anti-DNP antibody response to DNP-BSA was truly amplified by the secondary injection of DNP-HGG into mice primed with DNP-HGG, and that the cell-associated carrier-specific memory as judged by the helper effect on anti-DNP response to DNP-BSA was also truly amplified by the secondary injection of BSA into mice primed with BSA. However, when memory was assessed in actively immunized mice, the secondary injection of BSA into mice primed with DNP-BSA and HGG decreased anti-DNP responsiveness to the tertiary injection of DNP-BSA, whereas the secondary injection of DNP-HGG secondarily increased anti-DNP responsiveness. In mice primed with DNP-BSA the titers of serum antibodies to BSA increased after the secondary injection of DNP-BSA or BSA. From these results it has been concluded that, like B cell-associated memory, T cell-associated memory is also amplified by a secondary antigenic stimulus, although its expression is inhibited in actively immunized mice through negative control by their antibodies.     

Haptenation of adult Schistosoma mansoni and assessment of humorally mediated damage in vitro

Adult Schistosoma mansoni were haptenated with DNP under mild conditions using DNP-liposomes, as devised by Levi-Schaffer et al. (1983, Am. J. Trop. Med. Hyg. 32: 343) for haptenating schistosomula. The effects of complement, alone or with anti-DNP antibodies, on tegumental morphology of DNP-haptenated adult worms were assessed by both scanning and transmission electron microscopy, as a simple model system for possible in vitro effects of complement and anti-schistosomal antibodies on normal adult worms. Complement-mediated cytotoxicity, as measured by tegumental changes in both normal and haptenated worms, was observed and indicates a possible role for complement in humorally mediated damage of adult schistosomes. The damage to haptenated adults in the presence of both complement and anti-DNP was greater than that to nonhaptenated worms similarly treated. However, anti-DNP plus complement caused less damage to normal worms than did complement alone, perhaps the result of blocking by antibodies bound to the tegument via Fc receptors. Evidence herein presented implicates both classical and alternative complement activation by the adult worm's tegument. Tegumental tubercle smoothing was one manifestation of damage, as assessed by SEM. Smoothing appears to progress from spine blunting to disappearance, arguing for resorption rather than shedding as the mechanism for spine loss. Because the spines are mostly actin bundles, we suggest that complement-mediated intrategumental calcium ion flux could lead to spine resorption resulting from actin filament depolymerization and/or calcium-induced severing of actin filaments and dissociation of actin filament cross-linking proteins. This process could have adaptive value to the adult worm, in that the spines may be a repository of readily mobilizable actin molecules for tegumental maintenance and repair.     

Effect of Adjuvants on the Antibody Response to a Hapten on a Thymus-independent Carrier

IT has been demonstrated in mice that levan (polyfructose), an antigen which interacts only with B lymphocytes¹, can function as a carrier and produce a thymus-independent response to dinitrophenol (DNP)². Using this conjugate, antibody-production against the hapten is totally unaffected in thymectomized animals and is abolished in mice tolerant to levan. The DNP-levan conjugate produces only 19S antibody against DNP.     

Role of self carriers in the immune response and tolerance. X. A lymphoid dendritic-like tumor, P388AD.2, acts as a novel immunogenic carrier for hapten

Hapten-modified spleen cells, peritoneal exudate cells, and certain lymphoid tumors preferentially induce specific tolerance after i.v. administration. In contrast to these tolerogenic carrier cells, we found that a haptenated lymphoid dendritic-like tumor, P388AD.2, acts as a potent immunogen after i.v. injection. The immunogenicity of P388AD.2 was analyzed by measuring the specific augmentation of plaque-forming cell (PFC) responses when spleen cells from mice previously injected with haptenated tumor cells were challenged in vitro with thymus-independent antigens. Optimal immunization was found to be dependent on cell dose and hapten concentrations. Further studies indicated that P388AD.2 elicited a response which was T cell-dependent and which involved both the so-called Lyb-3,5,7- and Lyb-3,5,7+ B cell populations. Injection of haptenated tumor into different mouse strains suggested that H-2 compatibility was required to prime B cells in vivo, although significant augmentation could also be achieved in allogeneic C57B1/6J mice. The enhanced PFC responses elicited in H-2b mice could not be explained by allo-recognition of class I or II MHC determinants. In toto, these results suggest that P388AD.2 acts as a unique accessory cell for the presentation of hapten-modified self.     

Induction of in vivo helper activity for murine antibody responses by macrophages pulsed with ovalbumin-monomethoxypolyethylene glycol (OA-mPEG) conjugates

Conjugates of protein antigens with an optimal number of monomethoxypolyethylene glycol (mPEG) chains of an appropriate molecular weight had been shown to suppress murine IgE responses to the unmodified antigen. To investigate the possibility that the tolerogenic capacity of these mPEG conjugates is attributable to a defect in macrophage (M phi) presentation of their antigenic determinants, the activity of ovalbumin (OA)-mPEG conjugates when pulsed onto mouse peritoneal adherent cells (M phi) was compared in this study with their activity in solution. Surprisingly, in contrast to the suppressogenic capacity of mPEG conjugates in solution, the OA-mPEG pulsed M phi appeared to exert a helper effect when injected intraperitoneally (ip), i.e., after subsequent immunization with dinitrophenylated OA (DNP3-OA) in Al(OH)3, the mice showed accelerated IgE and IgG1 antibody responses to OA and DNP. However, when M phi were exposed to limiting concentrations of OA or OA-mPEG, markedly higher concentrations of OA-mPEG were required to yield pulsed M phi, exerting a significant helper effect. It was concluded that although M phi were capable of presenting the OA determinants of OA-mPEG conjugates to helper T (Th) cells, the preparations of modified antigen were presented less effectively than native OA.     

Induction of anti-hapten antibody response by hapten-isologous carrier conjugate. II. Specificity of hapten-reactive helper T cells.

Anti-hapten antibody production was elicited by the immunization of hapten-isologous carrier conjugate (PAB-MGG) in mice. Spleen and lymph node cells taken from these primed mice could demonstrate their helper activity for anti-DNP antibody production when transferred intravenously into 600R X-irradiated recipient mice along with DNP-primed B cells and the double hapten conjugated carrier, DNP-MGG-PAB. Isologous carrier (MGG)-primed cells could not demonstrate this helper activity. Accordingly, helper cells reactive for a haptenic group are considered to develop by the immunization of hapten-isologous carrier conjugate. Hapten-reactive helper activity was also induced by the immunization of other hapten-isologous carrier conjugates, e.g., MAB-MGG, PABS-MGG or PAB-MSA. These hapten-reactive helper cells were T lymphocytes, as the helper activity of PAB-MGG-primed cells was completely abolished by in vivo ATS-treatment. Helper activity of PAB-MGG-primed cells for DNP-primed B cells was also demonstrated through the double hapten conjugated heterologous carrier DNP-HGG-PAB to be the same as with DNP-MGG-PAB, but weakly through DNP-KLH-PAB. As HGG but not KLH resembles MGG in composition, almost all hapten-reactive helper T cells can be considered to recognize not only haptenic groups but also physicochemical properties of the hapten-conjugated carrier site. However, these helper T cells could discriminate structural differences among related haptenic groups, because PAB-MGG-primed cells clearly responded to DNP-MGG-PAB to demonstrate their helper activity for DNP-primed B cells, but responded only weakly to DNP-MGG-PABS or DNP-MGG-MAB. When the specificity restrictions of T and B cells to the same haptenic group were compared by responsiveness measured after the antigenic stimulation (B cell function by anti-hapten antibody production and T cell function by helper activity), differences were noted, as PAB-MGG-primed T cells could respond not only to DNP-MGG-PAB but also fairly well to DNP-MGG-MAB to demonstrate their helper activity, but PAB-MGG-primed B cells responded to only PAB-MGG. Thus, hapten specificity appears to be much more restricted for B cells than T cells. The difference of this responsivity between B cells and helper T cells was thought to derive from the specificity difference of B cell and helper T cell receptors rather than from any sensitivity differences of the experimental procedure. The differences in the specificity restrictions of receptors of B and helper T cells were discussed in the light of hapten-specificity.     

Induction of anti-hapten antibody response by hapten-isologous carrier conjugate. I. Development of hapten-reactive helper cells by hapten-isologous carrier

Anti-hapten antibody production was elicited by the immunization of hapten-isologous carrier conjugate (DNP-MγG) in mice. The spleen and lymph node cells taken from those primed mice were effectively stimulated with hapten-heterologous carrier conjugates (DNP-KLH and DNP-BαA) as well as hapten-homologous carrier conjugate (DNP-MγG) when transferred into X-irradiated recipient mice. The reactivity of DNP-MγG-primed cells to DNP-heterologous carrier conjugates was not due to the mutual crossreactivity of the carrier with MγG on cellular level, since the spleen and lymph node cells primed with DNP-KLH or DNP-BαA could only be stimulated with corresponding hapten-homologous carrier conjugate. The responsiveness of DNP-MγG-primed cells to hapten-heterologous carrier conjugates was due to the result that hapten-reactive helper cells were developed by the immunization of hapten-isologous carrier and these cells cooperated with hapten-specific B cells.The helper activity of the hapten-isologous carrier-primed cells was resistant to 600-R X-irradiation in vitro and sensitive to in vivo ATS treatment. This suggests that the helper activity induced by hapten-isologous carrier is of T cell origin. The helper activity of hapten-isologous carrier-primed cells was also developed by the immunization of PAB-MγG, and clear cooperative interaction between PAB-MγG-primed cells and DNP-specific B cells was demonstrated through DNP-MγG-PAB.The possible mechanism of helper cell development induced by the immunization of hapten-isologous carrier conjugate was discussed in light of the hapten specificity of helper activity.     

Localization of Coliphage MS2 A-Protein

The purification of coliphage MS2 dinitrophenol (DNP) conjugates provided a system for localization of the single molecule of A-protein in the capsid of the MS2 phage particle. Three A-protein preparations isolated from unconjugated MS2, overconjugated DNP-MS2, and purified 78S DNP-MS2 were tested for the presence of covalently bound DNP. The binding characteristics to Dowex 1-X8 and rabbit anti-DNP bovine serum albumin (DNP-BSA) immunoglobulin G of the 78S DNP-MS2 and overconjugated DNP-MS2 A-protein preparations indicate that the A-protein is located on the surface of the phage particle where it can be covalently conjugated with hapten. Extensive enzymatic iodination of the A-protein of intact unconjugated MS2 substantiates this conclusion.     

Interactions of antigen-sensitized liposomes with immobilized antibody: a homogeneous solid-phase immunoliposome assay

Dioleoyl phosphatidylethanolamine (DOPE) does not form stable bilayer liposomes at room temperature and neutral pH. However, stable unilamellar liposomes could be prepared by mixing DOPE with a minimum of 12% of a haptenated lipid, N-(dinitrophenylaminocaproyl)-phosphatidylethanolamine (DNP-cap-PE). When the liposomes bound to rabbit anti-DNP IgG that had been adsorbed on a glass surface, lysis of the liposome occurred with the release of the contents into the medium as judged by the fluorescence enhancement of an entrapped self-quenching dye, calcein. On the other hand, incubation of the same liposomes with glass surfaces coated with normal rabbit IgG had little effect. In addition, free anti-DNP IgG induced aggregation of the liposomes but did not cause any dye release. Liposomes composed of dioleoyl phosphatidylcholine (DOPC) and DNP-cap-PE did not lyse when added to the glass surfaces coated with either anti-DNP IgG or normal IgG. A likely mechanism for liposome lysis is that the DNP-cap-PE laterally diffuse to the contact area between the liposome and the glass. Binding of the haptenated lipid with the immobilized and multivalent antibody trap the haptenated lipids in the contact area. As a result of lateral phase separation, lipids may undergo the bilayer to hexagonal phase transition, leading to the leakage of the entrapped dye. Because both the free hapten and the free antibody inhibited the liposome leakage, this process could be used to assay for the free hapten or antibody. We have shown that inhibition assays performed by using this principle can easily detect 10 pmol of free DNP-glycine in 40 microliter. Furthermore, by substituting human glycophorin A, a transmembrane glycoprotein, for the lipid hapten, we have demonstrated that this assay system is also applicable to detect protein antigen with a sensitivity of sub-nanogram level.     

Tolerance defects in New Zealand Black and New Zealand Black X New Zealand White F1 mice

The susceptibility of autoimmune NZB and (NZB X NZW)F1 mice to the induction of tolerance by monomeric BSA was compared with several normal mouse strains. Unresponsiveness in T and B lymphocyte compartments was probed by challenging with DNP8BSA and measuring anti-DNP and anti-BSA antibodies separately. Tolerance induced by monomeric BSA was carrier specific, and there was no evidence of epitope-specific suppression. Normal NZW, NFS, and B10.D2 mice were easily rendered tolerant with monomeric BSA and did not produce anti-DNP or anti-BSA antibodies after challenge with DNP8BSA. By contrast, the lack of anti-DNP antibody response in similarly treated NZB mice was dependent on the dose of monomeric BSA, indicating that the helper T cells were partially resistant to tolerance induction. NZB mice treated with a high dose of monomeric BSA produced anti-BSA, but not anti-DNP, antibodies after immunization. Thus, the anti-carrier B cells in NZB mice may have been primed by monomeric BSA. The presence of the xid gene on the NZB background rendered the mice susceptible to induction of tolerance, suggesting that the tolerance defect in NZB mice involves the B cell compartment. This abnormal antibody response was a dominant trait: (NZB X NFS)F1 and (NZB X B10.D2)F1 mice had the same characteristics as NZB mice. These F1 hybrids do not develop autoimmune disease, indicating that resistance to experimental tolerance induction expressed at a B cell level may not be sufficient for disease development. In contrast to NZB and other NZB F1 hybrids, (NZB X NZW)F1 hybrids treated with monomeric BSA and challenged with DNP8BSA responded to both DNP and BSA. The contribution of a B cell defect to the tolerance abnormality of (NZB X NZW)F1 mice was examined by analyzing the effect of the xid gene on the progeny of (NZB.xid X NZW)F1 mice. Unlike the effect of the xid gene on NZB mice, both phenotypically normal heterozygous female and phenotypically xid hemizygous male mice produced anti-DNP and anti-BSA antibodies after tolerance induction and immunization, demonstrating that a major helper T cell abnormality was present in (NZB X NZW)F1 mice. The (NZW X B10.D2)F1 hybrid was rendered tolerant by this procedure, indicating that the helper T cell defect (NZB X NZW)F1 mice may have resulted from gene complementation with the NZB mice contributing partial resistance of T helper cells to tolerance induction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Contact hypersensitivity reactions to dinitrofluorobenzene mediated by monoclonal IgE anti-DNP antibodies

Several studies have suggested a possible role for IgE antibodies in the pathogenesis of cutaneous hypersensitivity reactions that reach maximum intensity 24 to 48 hr after antigen challenge. The recent availability of murine monoclonal IgE anti-hapten antibodies has made possible the direct examination of the range of cutaneous inflammatory reactions that can be mediated by such antibodies. We have examined the effects of passively sensitizing BALB/c mice with monoclonal IgE anti-dinitrophenyl (DNP) antibody 48 hr before antigen challenge. Inflammatory responses were assessed by measuring ear swelling in mice challenged on the ears with the reactive hapten 2,4-dinitrofluorobenzene (DNFB). Compared with unsensitized controls, the ears of mice passively sensitized with IgE anti-DNP displayed a biphasic pattern of ear swelling after DNFB challenge. An early, transient response (present within 15 to 30 min of challenge and returning to control levels within 4 to 9 hr) was followed by a second, more persistent increase in ear swelling that peaked 24 to 48 hr after challenge. This biphasic pattern of ear swelling seen in IgE-sensitized mice was temporally indistinguishable from that observed in mice conventionally sensitized for allergic contact dermatitis reactions by epicutaneous application of DNFB 5 days before DNFB ear challenge. Antigen specificity of the IgE-mediated contact hypersensitivity reactions was demonstrated by the failure of mice passively sensitized with IgE anti-DNP to display early or delayed ear swelling greater than unsensitized controls when challenged with either of two noncross-reacting haptens, fluorescein isothiocyanate or oxazolone. Mice passively sensitized with a monoclonal IgA anti-DNP antibody (MOPC 315) 48 hr before DNFB challenge failed to display early or delayed ear swelling greater than unsensitized controls. Heat inactivation of the IgE anti-DNP ascitic fluid at 56 degrees C for 30 min completely abolished its capacity to passively sensitize mice for contact hypersensitivity reactions after DNFB challenge. These results document the existence of an antigen-specific, IgE-mediated, delayed-in-time cutaneous hypersensitivity response that can be elicited by epicutaneous challenge (contract) with a reactive hapten.