Hapten-specific T-cell lines in guinea-pig contact sensitivity: carrier specificity and specific accumulation.

Dinitrophenyl-specific T-cell lines were established by culturing lymph node-derived lymphocytes from dinitrochlorobenzene-sensitized guinea-pigs with dinitrophenyl-modified macrophages. Cells from this line were all Ia-positive and formed E-rosettes with rabbit erythrocytes; no Ig-positive cells were present in this suspension. Expanded cells exhibited enhanced in vitro and in vivo activity as demonstrated by DNA synthesis and systemic adoptive transfer of contact sensitivity. In vitro DNA synthesis was elicited not only be specific hapten-modified macrophages but also by conjugates of the hapten with homologous (GPA) and even heterologous (BGG, HSA) proteins, thus demonstrating the loss of carrier specificity. Moreover, the proliferative response of expanded cells was elicited not only with hapten-modified syngeneic (strain 2) but also with allogeneic (strain 13) macrophages. Cells from hapten-modified T-cell lines, but not from lymph nodes from in vivo primed guinea-pigs, showed specific accumulation in contact sensitivity skin test sites. Attempts to establish hapten-specific T-cell clones were also at least partially successful.

Antigenic competition in the induction of contact sensitivity in the guinea pig.

The antigen-presenting capability of macrophage modified with a single hapten (dinitrofluorobenzene) is partially or totally abolished by an additional haptenation with a second related (picryl chloride) or unrelated (oxazolone) hapten. This effect, 'antigenic competition', is only partially mediated by suppressor cells. There also seems to be an inhibition of the association of the hapten with particular Ia antigens. The prerequisite for antigenic competition is that both hapten responses are controlled by the same immune response gene. Hapten responses which are controlled by different genes, e.g., dinitrofluorobenzene, picryl chloride, and oxazolone on the one hand and benzilidene acetone on the other, do not compete. The pattern of competition thus varies with the strain of guinea pig.

Induction of contact sensitivity to a broad variety of allergens with haptenized macrophages.

Using guinea pigs an analysis could be made of various aspects of contact sensitivity (CS) induced by subcutaneous injection of syngeneic haptenized macrophages (oil-induced peritoneal exudate cells, PEC) as compared to epicutaneous sensitization. Very little PEC-bound hapten (dinitrochlorobenzene, or oxazolone) is needed for optimum sensitization. Nevertheless, both sensitization methods induce a state of CS that may last for over 6 months, give rise to hapten-specific antibodies with a similar isotype distribution, and show susceptibility to cyclophosphamide pretreatment. In addition, time courses and microscopic appearance of skin test reactions after either way of sensitization are identical. CS to a broad variety of physicochemically different antigens, including nickel, penicillin, and acrylates, is readily induced by syngeneic PEC, haptenized following a standardized procedure. As Freund's complete adjuvant is known to cause serious side effects like ulceration and long-lasting granuloma formation, immunization with haptenized PEC should now be considered as a clean and effective alternative in experimental CS studies.

Contact sensitivity in guinea pigs: allogeneic reaction and carrier specificity in antigenic recognition.

in the present paper it is shown that both induction and elicitation of 2,4-dinitrochlorobenzene-contact sensitivity in guinea pigs are genetically controlled. This genetic control is operating by two somehow different mechanisms. The recognition of hapten on allogeneic macrophages during the inductive phase is suppressed by the allogeneic response against these macrophages whereas the elicitation upon transfer of syngeneically primed lymphocytes is limited by the identity of at least some components of the major histocompatibility complex. This nonidentity contradicts the rule of carrier specificity, which is a requirement for all delayed type hypersensitivity reactions. Furthermore, the presentation of haptens to primed cells is less restricted than presentation of protein antigens.

Antigen-specific T-cell lines in DNCB-contact sensitivity in guinea pigs.

Expanded DNP-specific guinea pig T cells exhibit enhanced in vitro and in vivo activity as demonstrated by DNA synthesis and systemic adoptive transfer of contact sensitivity. Moreover, expanded lymphocytes are capable of accomplishing local passive transfer of contact sensitivity and producing interleukin 2, vascular permeability-increasing and macrophage migration-inhibiting factors. Expansion of hapten-specific lymphocytes offers a suitable model for studying eliciting mechanism of allergic contact dermatitis in humans.

[Genetic control of contact hypersensitivity in guinea pigs]. / Genetische Kontrolle bei der Kontaktüberemfindlichkeit des Meerschweinchens.

Guinea pigs sensitized intradermally with haptenized macrophages exhibit a positive skin reaction to an epicutaneous challenge with the specific hapten only when syngenic macrophages were used. Skin reactions to the hapten were in guinea pigs treated with haptenized allogenic macrophages always negative. There are two possible explanations: (1) T-lymphocytes are unable to recognize the hapten on allogenic macrophages, and consequently the animal is not hypersensitive. (2) The antigenic complex used for sensitization (haptenized allogenic macrophages) is not identical with the complex formed during the epicutaneous challenge with the hapten in respect of the carrier. Therefore, the skin reaction to the hapten remains negative.

Differential capacity of macrophages from various sources to act as hapten-specific stimulator cells in vitro.

Lymphocytes from 2,4-dinitrochlorobenzene, contact sensitized guinea pigs show increased DNA synthesis in vitro when stimulated by dinitrophenylated macrophages. In this study, macrophage-containing cell suspensions were isolated from various sources (spleen, lungs, peritoneal cavity) and from the peritoneal cavity also after different stimuli (oil, thioglycollate, starch, lymphokines). These cells were then haptenized and investigated on their capacity to act as stimulator cells in vitro. In addition, a possible relationship between Ia-positivity of the hapten-presenting cell suspensions and the induction of DNA synthesis was studied. The results demonstrate (1) that the hapten-presenting capacity of macrophages differs with respect to the source and the way of elicitation, (2) that oil-induced peritoneal exudate cells are the most suitable stimulator cells for in vitro assays in contact sensitivity, and (3) that the cellular expression of Ia antigens is in itself no warrant for hapten-presenting capacity.

Self molecules in induction of hypersensitivity and tolerance in DNCB contact sensitivity in the guinea pig.

Dinitrophenylated macrophages are efficient inducers of contact sensitivity and of tolerance to dinitrochlorobenzene, depending on the mode of application. Contact sensitivity induced by an intradermal injection of DNP-M phi is genetically restricted, whereas tolerance induced by an intravenous injection is, in guinea pigs, not under control. This tolerance is complete in the majority of animals, but is reversed by DNCB in Freund's complete adjuvant and prevented by pretreatment with cyclophosphamide. Tolerance induced by an intravenous injection of the unconjugated hapten is not reversed by DNCB in FCA, but is abrogated by an intradermal injection of DNP-M phi. This abrogation does not occur, however, when DNCB in FCA is applied simultaneously with the DNP-M phi. In further experiments, it is demonstrated that the genetic restriction of induction of contact sensitivity by DNP-M phi is on the level of antigen recognition and is not due to the nonidentity of the Ia structures on macrohages used for induction and the Ia structures involved in elicitation of contact sensitivity.

Characterization of lymphocyte subpopulations which exhibit enhanced DNA synthesis in vitro in DNFB contact sensitive guinea-pigs.

Different lymphocyte subpopulations were prepared from lymph nodes and spleen obtained from 2.4-dinitrofluorobenzene/Freund's complete adjuvant (DNFB/FCA) sensitized guinea-pigs. The capacity to react in vitro to secondary antigenic (dinitrophenyl-modified syngeneic macrophages, DNP-MO) stimulation by an increased DNA synthesis, appeared to be restricted to those T cells which lack receptors for IgG-Fc (T gamma -). We confirmed the existence in this species of an IgG-Fc receptor-positive T-cell subset, which is particularly represented in the spleen (up to 56%). These T gamma + cells apparently are not of monocyte lineage as they show strong PHA-responsiveness and a lack of non-specific esterase staining, whereas the majority of these cells form RRBC-rosettes. Neither B nor T gamma + cells appeared to act as suppressor cells in the secondary DNP-MO-driven proliferative response.

In vitro DNA synthesis in lymphocytes from guinea pigs epicutaneously sensitized with DNCB.

Lymph node lymphocytes from guinea pigs sensitized with the hapten homogenized in Freund's complete adjuvant or pretreated with cyclophosphamide shortly before an epicutaneous sensitization are capable of responding in vitro to a larger set of antigenic preparations than the lymphocytes from only epicutaneously sensitized animals. This may be due to a formation of a larger set of antigenic determinants when the hapten was homogenized in Freund's complete adjuvant or to a formation of a larger repertoire of lymphocytes capable of recognizing these determinants as it might be the result of pretreatment with cyclophosphamide. These procedures may also change the proportion of responding cells in the lymphocyte suspensions.

In vitro DNA synthesis in lymphocytes from DNCB contact-sensitive guinea pigs.

In vitro stimulation of lymphocytes from DNCB contact-sensitive guinea pigs is achieved by several DNP conjugates. Optimal results are obtained by DNP-modified macrophages. The degree of in vitro lymphocyte activity, demonstrated by DNA synthesis, depends on the cell origin, the interval between sensitization and harvest of lymphocytes and the immunological status of the animals.

Recent trends in the immunology of contact sensitivity. I.

The article reviews the recent experimental data that influenced the present concept of contact sensitivity. Particular emphasis is on the mechanism of stimulation of antigen-inexperienced specific lymphocytes, the eliciting phase, and the role of suppressor cells in sensitization and tolerance.

Mechanism of densensitization in DNCB-contact sensitive guinea pigs.

Contact sensitivity to dinitrochlorobenzene (DNCB) in guinea pigs could be rapidly suppressed by intravenous injection of dinitrobenzene sulfonic acid sodium salt (DNBSO3). This suppression is transient and antigen-specific. Macrophages from desensitized animals are not inactivated as shown by their ability to react, both in vivo and in vitro to lymphokines produced in a separate system. Therefore, effector lymphocytes are considered the target for the desensitizing antigen. Using an adoptive transfer system it was demonstrated that effector lymphocytes are inactivated by a direct effect of the hapten. Since this inactivation can be reversed by trypsin treatment, a receptor blockade of effector lymphocytes is proposed as the mechanism of desensitization of DNCB-contact sensitive guinea pigs. This does not exclude the possibility that additional mechanisms such as suppressor cells, compartmentalization or endogenous proliferation of lymph node lymphocytes may play an additional role.