Scratching behavior in spontaneous- or allergic contact-induced dermatitis in NC/Nga mice.

NC/Nga mice have pathological and behavioral features similar to those seen in human atopic dermatitis. There are two known dermatitis models in NC/Nga mice, one being spontaneous-induced dermatitis under conventional conditions and the other 2,4,6-trinitrochlorobenzene (TNCB)-induced allergic contact dermatitis. However, there are significant differences in time course on development of dermatitis. We studied the role of scratching behavior (sign of itch) on the development of dermatitis on spontaneous- and TNCB-induced dermatitis. We measured scratching counts, transepidermal water loss (TEWL), and skin inflammation score, under conventional conditions or by applying 5% TNCB once a week for 6 weeks in NC/Nga mice. In spontaneous-induced dermatitis, scratching counts increased with the passage of time. The scratching counts were significantly increased only 1 week after housing the mice under conventional conditions, but no changes were observed in cases of TNCB-induced dermatitis. In spontaneous-induced dermatitis, TEWL and skin-inflammation score were gradually increased, time-dependently. On the other hand, in TNCB-induced dermatitis, these dependent values rapidly increased and reached a maximum only after 24 h TNCB application. These data suggest that pathogenesis of spontaneous- and allergic contact-induced dermatitis was clearly different. It will be of major interest to identify the pruritic mediators causing profound scratching behavior and scratching-induced aggravation of inflammation in the spontaneous-induced dermatitis, as opposed to the inflammatory mediators that cause contact allergic dermatitis without major scratching.

Canonical germinal center B cells may not dominate the memory response to antigenic challenge.

Spleen and bone marrow (BM) are the major sites of antibody production and anamnestic response in systemically immunized mice. We examined the VDJ segment repertoire of antibody plaque-forming cells (APFC) in those two sites in the course of antibody responses to the hapten nitrophenyl (NP). Individual IgG APFC expressed any one of 10 V(H) segments of the V186.2/V3 (J558) gene family: 186.2, 102, 23, C1H4, 165.l, CH10, 3, 593.3, 24.8 and 671.5. The majority of cells in both spleen and BM expressed the V186.2 gene joined to a D segment with Tyr95. During a 2-month period after a single immunization, the V186.2(+) APFC in BM accumulated 3 times as many somatic mutations than splenic APFC (average 8.5 versus 3 mutations/V(H)); this process was T(h) dependent as shown by in vivo depletion of CD4(+) lymphocytes. However, the V186.2(+) APFC in both spleen and BM shared a recurrent W33L replacement, indicating their common origin from germinal centers. The APFC expressing the other (analogue) V(H) segments were evenly represented in the spleen and BM, but they accumulated few, if any, mutations. The anamnestic V186.2(+) APFC were highly mutated both in the spleen and BM; they represented a new and unexpected clonotype. The V/D segments were joined by Gly95 instead of Tyr95, the W33L was absent and a new shared K58R replacement appeared. The APFC expressing the 'analogue' V(H) genes comprised approximately 20% of the anamnestic response and did not accumulate more mutations, but their affinities were in the range of the memory V186.2(+) cells. These data suggest that the late primary and secondary responses to a hapten may be born by different B cell lineages, and that some clonotypes may reach the memory pool without an extensive mutation and expansion.

Exploitation of a monoclonal antibody for weak affinity-based separation in capillary gel electrophoresis.

Weak biospecific recognition has been established for affinity separation in high performance liquid chromatography (HPLC). The use of weak affinity chromatography (WAC) has been limited previously by the insufficient separation efficiency achieved, allowing only some 1000 plates/m to be obtained. However, it has been shown that chiral drug separation can be performed with capillary affinity gel electrophoresis (CAGE) at considerably improved efficiency as compared with traditional chromatographic procedures. Our present study demonstrates the potential of weak affinity monoclonal antibodies as a generic method for immunologically based separations in capillary electrophoresis. Monoclonal antibodies were polymerized within a silica capillary and were used for the separation of structurally similar carbohydrate antigens. The results indicate that weak biospecific interactions can be utilized in a CAGE format to produce highly selective separation of the alpha- and beta-forms of p-nitrophenyl-labeled maltose. It remains to be seen, however, how efficient weak affinity separation in CAGE can be compared with affinity HPLC protocols. Details of typical separations and of the preparation of the antibody gel are presented.

Contact allergens and epidermal proinflammatory cytokines modulate Langerhans cell E-cadherin expression in situ.

After exposure to antigen, Langerhans cells (LC) migrate from the epidermis to lymph nodes, where they initiate primary immune responses in T cells. The adhesion molecule E-cadherin mediates adhesion of LC to keratinocytes in vitro and may be responsible for localization of LC in epidermis. To determine if levels of LC E-cadherin are modulated during LC emigration from epidermis, we utilized flow cytometry to evaluate E-cadherin expression on BALB/c LC exposed in situ to the contact allergen 2,4,6-trinitrochlorobenzene (TNCB). TNCB induced increased I-A/E antigen and decreased E-cadherin expression on a subpopulation of LC as early as 12 h, and as late as 48 h, after application. At 24 h, approximately 30% of LC in TNCB-treated skin expressed increased I-A/E antigens; of these activated LC, approximately 40% expressed decreased levels of E-cadherin. E-cadherin levels on this latter subset were approximately 15% of those expressed by LC in normal skin, and were similar to levels on cultured LC and LC that migrated from skin explants. The effect was specific for allergens; no changes occurred in LC following treatment with several contact irritants or the tolerogen dinitrothiocyanobenzene. To determine if cytokines modulated LC E-cadherin expression, we introduced various cytokines into BALB/c ear skin and assayed I-A/E antigen and E-cadherin levels. Of the cytokines tested, only interleukin-1 and tumor necrosis factor alpha reproduced the effects of TNCB. We propose that downmodulation of E-cadherin expression occurs as a consequence of local cytokine production during antigen-induced LC activation, facilitating LC emigration and the initiation of immune responses against antigens encountered in epidermis.

Lipopolysaccharide from Brucella abortus behaves as a T-cell-independent type 1 carrier in murine antigen-specific antibody responses.

In order to determine the carrier nature of lipopolysaccharide from Brucella abortus (LPS-BA) in evoking humoral responses, normal and immunodeficient mice were immunized with trinitrophenyl (TNP)-conjugated LPS-BA (TNP-LPS-BA) and the responses were compared with those to known T-dependent and T-independent antigens. TNP-LPS-BA, like T-independent type 1 (TI-1) antigens such as TNP-BA and TNP-LPS from Escherichia coli (TNP-LPS-EC), generated anti-TNP responses in BALB/c, athymic BALB/c nu/nu, and CBA/N mice. In contrast, N-2,4-dinitrophenyl-beta-alanylglycylglycyl-substituted keyhole limpet hemocyanin, a typical T-dependent antigen, was not immunogenic in athymic mice, and TNP-Ficoll (T-independent type 2) was ineffective in eliciting humoral responses in CBA/N mice. These results indicate that LPS from B. abortus acts as a TI-1 carrier in generating antibody responses. In C3H/HeJ mice, TNP-LPS-BA generated higher-titer immunoglobulin G1 (IgG1), IgG2a, and IgG2b anti-TNP antibodies than TNP-LPS-EC. Compared with those from BALB/c mice, pure resting B cells isolated from C3H/HeJ mice exhibited a 30-fold lower proliferative response to LPS-EC, whereas the LPS-BA response was reduced to a lesser extent (5-fold). This suggests that the disparity observed in antibody titers was due to different abilities of LPS from B. abortus and E. coli to stimulate C3H/HeJ B cells. The ability of LPS from B. abortus to act as a carrier in generating humoral immune responses indicates that LPS-BA can be substituted for whole B. abortus organisms in vaccine development.

Human IgG subclass pattern of inducing complement-mediated cytolysis depends on antigen concentration and to a lesser extent on epitope patchiness, antibody affinity and complement concentration.

The relative complement-mediated lytic capability of the IgG subclass isotypes was studied using a matched set of mouse-human chimeric anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies. The subclass pattern was shown to be highly dependent on variations in antigen concentration and to lesser extent on variation in epitope patchiness, antibody binding affinity and complement concentration. In general, the IgG3 subclass was most effective in inducing cytolysis at the different conditions used and only at high antigen concentration did the IgG1 subclass mediated more efficient cytolysis than IgG3. The IgG2 isotype required a relative high antigen concentration to be cytolytic while the IgG4 isotype was not cytolytic at any of the conditions tested. These individual characters of each of the IgG subclasses makes it conceivable that a subtle system of immunoregulation exists among the subclasses.

Thymus-dependent antiidiotype and anti-antiidiotype responses to a dinitrophenyl-specific monoclonal antibody.

BALB/c mice were inoculated i.p. with graded doses of a DNP-specific, IgM mAb (designated 57.1). Injection with unmodified 57.1 in the absence of adjuvants resulted in the generation of an anti-Id response (Ab2) and an anti-anti-Id response (Ab3). The generation of serum anti-Id antibodies was found to be thymus dependent. Nude mice immunized with 57.1 were unable to produce a serum Ab2 response above nonimmunized controls whereas euthymic mice receiving identical doses of 57.1 produced strong Ab2 responses. To examine the specificity of serum anti-Id, sera from mice receiving 57.1 were screened against a panel of mAb representing at least five distinct VH gene families. Serum titers were significantly higher against 57.1 than against any of the other antibodies in the panel. Three of the antibodies in this panel bind FD5-1, a monoclonal anti-Id (Ab2) that also binds 57.1. However, sera from mice receiving 57.1 bound 57.1 only. Thus, the serum Ab2 response appears to be highly specific for idiotopes on 57.1. The predominant isotype of these anti-Id antibodies was IgG1. The number of isotypes detected increased in a dose dependent manner with all IgG subclasses having anti-Id specificity in sera from animals receiving the higher doses of 57.1. Further analysis of the serum demonstrated that approximately 8% of the Ab2 response was paratope-specific (inhibitable by the monovalent hapten DNP-lysine). The same sera were analyzed for the presence of Ab3 by binding to the monoclonal anti-Id antibody FD5-1. Lower serum titers of Ab3 were generated in comparison to serum titers of Ab2. Analysis of the binding specificity of the Ab3 response revealed that DNP-BSA was able to partially inhibit the binding of serum IgM and IgG Ab3 to FD5-1. A subset of the Ab3 response. Ab1' that is specific for DNP was observed in a direct binding assay where detectable amounts of DNP binding IgM, IgG1, and IgG3 isotypes were present. We have thus described a complete circuit (Ab1----Ab2----Ab3) of antibodies within the Id network by immunizing animals with an unmodified mAb in the absence of Ag or adjuvants.

Alteration of idiotypic connectivity in prenatally tolerized neonatal mice.

Prenatal tolerization with trinitrobenzenesulphonic acid (TNBS) leads to expansion of trinitrophenyl (TNP)-specific B cells, the majority of which become refractory to stimulation during postnatal development. One possible explanation could be that they belong to the repertoire of naturally activated B cells which are limited in expansion after antigenic stimulation due to a high degree of idiotypic connectivity. To evaluate this hypothesis, 59 thymus- and 490 spleen-derived B-cell hybridomas from 6-day-old prenatally untreated and prenatally TNBS-treated mice were tested for reactivity against 33 arbitrarily chosen clones derived from the same fusions, 17 being derived from control and 16 from tolerized litters. Two major points could be deduced: (1) Idiotypic connectivity, including connectivity of TNP- and anti-TNP-reactive monoclonal antibodies (MoAb), was maintained after prenatal tolerization. This accounted for thymus- and spleen-derived MoAb. (2) Only TNP- and anti-TNP-reactive MoAb derived from prenatally untreated and prenatally tolerized mice displayed significantly distinct idiotypic profiles. Differences were pronounced, especially with thymus-derived MoAb. Thus, TNP-specific B cells in prenatally tolerized newborns do not behave like B cells of adult mice stimulated by external antigen, but rather like a part of the naturally activated, idiotypically connected B-cell repertoire of the newborn. This could explain B-cell unresponsiveness at older age as a consequence--at least partly--of their high idiotypic connectivity.

Anti-TNP antibody localization of the reactive lysine residues in myosin.

Myosin contains reactive lysine residues which are trinitrophenylated by 2,4,6-trinitrobenzene sulfonate much faster than the rest of the lysines. Here we find the location of these residues in the primary and spatial structure of myosin with the help of an anti-trinitrophenyl antibody. This antibody was raised against trinitrophenyl hemocyanin in rabbits. It reacted with trinitrophenylated myosin, and with some of the tryptic fragments of trinitrophenylated myosin. By analyzing the reaction with Western blots, it was found that the antibody preferentially reacts with the 27 kDa N-terminal fragment of the myosin head, and more weakly with the light meromyosin region of the myosin rod. The 27 kDa fragment contains the most reactive lysine residue, while the intermediate lysine residue is located in the light meromyosin region. The locations of the epitopes of the antibody were visualized on electron microscope images of rotary-shadowed trinitrophenylated myosin-antibody complexes. The distances of the epitopes to the head-rod junction of myosin were measured as 13 and 113 nm for the epitope on the head (reactive lysine residue) and for that on the rod (intermediary reactive lysine residue), respectively.

Anaphylactic properties of monohaptenic dinitrophenylated tripalmitoyl-S-glyceryl-cysteinyl lipopeptides.

Tripalmitoyl-S-glycerylcysteinyl lipopeptides are B-cell and macrophage activating and may be used as low molecular weight immunogens of considerable potency and even as vaccines when conjugated with suitable epitopic structures. Selected lipopeptides carrying single Dnp haptens were found to evoke mild passive cutaneous anaphylaxis in guinea pigs sensitized against Dnp. The reactions were observed after intravenous injection whereas intradermally applied antigen was negative. The anaphylactogenicity seems unrelated to micelle or aggregate formation of the insoluble peptides which require lecithin additions as well as sonication to become solubilized. The dinitrophenylated lipopeptide tripalmitoyl-S-glyceryl-cysteinyl-seryl-lysine produced toxic reactions which were not observed with the lipopeptide devoid of Dnp. Dinitrophenylated tripalmitoyl-S-glycerylcysteiny-1,6-diaminohexane and tripalmitoyl-S-glyceryl-cysteinyl-lysine did not show these toxic reactions.

Intrathymic T cell repertoire after prenatal trinitrobenzene-sulfonic acid-treatment.

It is still a matter of debate, whether tolerance toward self-non-MHC antigens is due to intrathymic deletion or to regulatory processes in the periphery. To further pursue this question, responsiveness toward TNP and an anti-TNP monoclonal antibody (Sp6) carrying a recurrent idiotype was evaluated in prenatally trinitrobenzenesulfonic acid (TNBS)-treated mice. In prenatally untreated as well as in TNBS-treated mice, thymocytes proliferating in the absence of nominal antigen were double negative (L3T4-/Lyt2-), but antigen-specific thymocytes were single positive (L3T4+/Lyt2- or L3T4-/Lyt2+). TNBS-treated mice differed from controls inasmuch as in their first week of life T cells proliferating in response to TNP were found in the thymus and detected at increased frequencies in the spleen. The frequency of TNP-specific thymocytes and spleen cells declined rapidly, finally reaching in the spleen a level of 20-30% of controls. Furthermore, after antigenic stimulation, the frequency of thymocytes and spleen cells proliferating in response to TNP was found to be increased in control mice, but TNP-specific T cell were no more recovered in the thymus or the spleen of tolerized mice. The same accounted for thymic and splenic T cells proliferating in response to Sp6. They were expanded in control mice after antigenic stimulation, but were undetectable in TNBS-treated mice. Thus, T cells with specificity for an internal (Sp6) and an external (TNP) antigen, provided the latter was present during ontogeny, were detected in the thymus of control and, transiently, in the thymus of tolerized mice. But, the fate of antigen-specific thymocytes was different in prenatally untreated and TNBS-treated mice. The data are interpreted in the sense that tolerance toward non-MHC antigens may be acquired subsequently to tolerance toward self-MHC antigens and possibly after imprinting of antigen specificity.

Influence of glutathione conjugation on the immunogenicity of dinitrophenyl derivatives in the rat.

The role of metabolic detoxication (glutathione conjugation) in the humoral response to three model haptens, dinitrofluorobenzene (DNFB), dinitrochlorobenzene (DNCB) and dinitrobenzenesulphonate (DNBS), was investigated in male Wistar rats. All three haptens produced a measurable anti-dinitrophenyl IgG antibody response over a wide dose range (2.7 nmol/kg to 0.27 mmol/kg) given for 4 days. A significant difference in antibody titre was only observed at the highest dose (DNFB greater than DNCB = DNBS), despite a marked difference in reactivity towards a protein carrier (albumin) and N-acetyl-lysine in vitro. These observations can be partly explained by the fact that the most reactive hapten (DNFB) conjugates most rapidly with glutathione in vitro and in vivo.

The specific interaction of helper T cells and antigen-presenting B cells. IV. Membrane and cytoskeletal reorganizations in the bound T cell as a function of antigen dose.

We have used double-immunofluorescence labeling to determine the surface distributions of LFA-1 and CD4, and the intracellular distributions of the cytoskeletal protein talin and of the microtubule organizing center (MTOC) of cloned Th cells in 1:1 cell couples with antigen (Ag)-specific APC of the B cell type (B-APC). The Th cell was directed to a peptide fragment of the Ag OVA in the context of IAd. The B-APC was the transfected A20 B hybridoma cell A20-HL, bearing on its surface a surface Ig specific for the hapten TNP, and pulsed with different concentrations of DNP-OVA. At sufficiently high doses of DNP-OVA (greater than 100 ng/ml), in essentially all couples, LFA-1, CD4, and talin were each concentrated at the Th cell membrane where it was in contact with the B-APC, and the MTOC inside the Th cell was reoriented to face the contact region. At lower doses of DNP-OVA (between 50 and 10 ng/ml), in all couples, LFA-1 and talin were concentrated at the Th/B-APC contact region, but the extent of CD4 clustering, MTOC reorientation, and Th cell proliferation all decreased with decreasing Ag dose. With no Ag, none of these effects was observed. These and other data indicate that two distinct signals are received by the Th cell that is specifically bound to its B-APC. The first signal, at low Ag doses, stimulates a linkage of LFA-1 and talin in the Th cell, and a specific LFA-1-mediated intercellular adhesion; the second signal, at higher Ag doses, is required to induce Th cell proliferation, with which the Th-MTOC reorientation and CD4 clustering are correlated.

Production and characterization of T-cell clones specific for trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self).

Using serial antigenic challenge as the method of selection and stimulation, continuous lines of cytotoxic T-lymphocytes (CTL) directed against TNBS-modified syngeneic spleen cells (TNP-self) have been generated. Spleen cells from C3H/HeJ (H-2k) mice were primed in vitro with autologous spleen cells modified with TNBS, and subsequently cloned by limiting dilution and in soft agar in the presence of IL2. These CTL clones grew continuously in medium supplemented with IL2 and in the presence of antigen. They are antigen specific and H-2 restricted in their target cell recognition. They all express Thyl and Lyt2 surface markers. None of the clones exhibit natural killer (NK) cell activity. All CTL clones tested so far are restricted in their target cell recognition to H-2Kk-TNP and none were found to be restricted to H-2Dk-TNP. These findings demonstrate at the clonal level the H-2K/D restriction of TNP-self specific CTL. These clones provide tools that may facilitate an understanding of the development and regulation of antigen specific CTL. They may also serve as models useful towards an understanding of the mechanism of lysis by CTL.

Antigen-specific inhibition of IL-2 and IL-3 production in contact sensitivity to TNP.

The production of IL-2 and IL-3 by T cells from mice which had been contact sensitized to TNP and/or tolerized by intravenous injections of TNBS was assayed. Contact sensitization rapidly primes T cells, so that they respond to in vitro restimulation with haptenated syngeneic cells by producing IL-2 and IL-3. This production is strongly inhibited, in an antigen-specific manner, in tolerized mice. At least part of this inhibition can be attributed to the action of suppressor T cells that act by preventing the activation of lymphokine production in vitro. Lymphokine production thus closely parallels the in vivo delayed-type hypersensitivity (DTH) reaction in this system.

Hierarchy of cytotoxic T cell clones. I. Reversal of resistance to lysis and killing between anti-hapten cytotoxic T cells.

Cells from clones of anti-hapten cytotoxic T lymphocytes (CTL) can act as both effector cells and, when treated with the specific hapten, as target cells. Individual clones can kill haptenated cells only from other clones that are less efficient killers. Clones specific for both fluorescein and trinitrophenol could be ordered in a single hierarchy in which resistance to lysis correlated with lytic efficiency. When the killing efficiency was reduced with phorbol myristate acetate (PMA) or the colchicine analogue, Colcemid, the degree of resistance to lysis was also reduced. The use of PMA-treated fluoresceinated targets greatly enhanced intraclonal killing and similarly lead to a repositioning of clones within the hierarchy of normal cells. By the haptenation of appropriate clones, efficient CTL could kill cells from other clones in a direction apparently opposite to recognition. The results demonstrate that effects other than antigen recognition of the target cell may result in variations in the nature of T cell immune responses.

Marginal zone of the murine spleen in autotransplants: functional and histological observations in the response against a thymus-independent type 2 antigen.

Splenic tissue from mice was autotransplanted; after initial necrosis, a rapid restoration of implants into a structure histologically indistinguishable from splenic tissue was observed. The development of the marginal zone in these autotransplants, as determined with monoclonal antibodies against different splenic cell types and routine histological stains, was compared with the local and systemic response against a thymus-independent (TI) type 2 antigen. Full restoration of time course and peak of anti-trinitrophenyl (TNP) serum titres against TNP-Ficoll was observed at 4 weeks after autotransplantation. Anti-TNP antibody-forming cells were observed in subnormal and normal numbers in 2- and 4-week old autotransplants, respectively. The appearance of normal numbers of antibody-forming cells, and the restoration of antibody titres at week 4 correlated with the return of newly formed B cells in a normal marginal zone. An unexpected observation was that marginal zone macrophages did not return until 10 weeks after transplantation, thereby making the necessity for these cells in the normal TI-2 response unlikely. We conclude that normal anti-TI-2 responses (onset and peak titres) can be restored by autotransplantation of splenic tissue. B cells and marginal zone organization are responsible for this response, for which marginal zone macrophages seem expendable. The partial protection against overwhelming post-splenectomy infections, given by autotransplants, can thus be explained by restorative capabilities of these implants on antigen presentation and antibody formation against TI-2 antigens, and not by an increase (compared with splenectomized individuals) of phagocytosis by marginal zone macrophages.

Studies on active immunization with self antigens. II. Production of antibody related to hapten substitution.

BALB/c mice were injected during neonatal life with conjugates in buffered physiological saline, prepared by coupling trinitrophenyl groups (TNP) at various densities to either syngeneic mouse serum albumin (TNP-MSA) or xenogeneic bovine serum albumin (TNP-BSA). Serum samples were obtained on days 30 and 60 after birth, on days 75 and 88 after two booster injections, and monoclonal antibodies were prepared from spleens of neonatally treated mice. The antibody titres, isotypes, and specificities were evaluated by enzyme-immunoassay. It was found that the extent of the anti-TNP immune response to TNP-MSA conjugates depends on the degree of hapten substitution, which is not the case for the anti-TNP-BSA. All the TNP-MSA conjugates induced mainly IgG and only a few IgM antibodies. These antibodies reacted essentially with the TNP group but seemed to have a higher avidity for the TNP-protein conjugate used in their induction. During the course of the immunization, decreasing quantities of TNP-MSA conjugates were needed to inhibit antibody binding. A large amount of monoclonal anti-TNP antibodies was found in hybridomas obtained after neonatal treatment either with TNP-MSA or TNP-BSA. Therefore, it appears that the anti-TNP immune response obtained after antigenic stimulation with sufficiently substituted TNP-MSA conjugates possesses all the characteristics of a normally occurring humoral immune response.