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Previous studies of cutaneous T cell-mediated responses in mice have obtained pharmacologic, morphologic, and immunologic evidence pointing to a critical role for local mast cells in release of the vasoactive amine serotonin (5-HT) to mediate early, initiating events that are required for elicitation of these responses. However, the role of mast cells in initiating these T cell-mediated cutaneous responses has been questioned due to the presence of relatively intact delayed-type hypersensitivity responses, such as contact sensitivity (CS), in mast cell-deficient mice whose skin contains only 1 % normal mast cell numbers. The contribution of other potential local sources of 5-HT, such as circulating platelets, at the site of a delayed-type hypersensitivity or CS response in these mast cell-deficient strains, has not been investigated. Therefore, we studied the effect of systemic platelet depletion, produced with an anti-platelet Ab, on blood and tissue levels of 5-HT, and on in vivo T cell-mediated cutaneous sensitivity responses, in W/Wv and Sl/Sld mast cell-deficient mice. The results showed that: 1) platelet depletion severely reduced whole blood 5-HT; 2) tissue levels of 5-HT, in mast cell-deficient mice, depended in large part on the presence of circulating platelets, and 3) specific depletion of platelets markedly suppressed CS responses in both W/Wv and Sl/Sld mast cell-deficient mice, and only moderately reduced CS in normal +/+ congenic mast cell-sufficient controls, but did not decrease CS in beige mice, with platelet granules that are defective in storage of 5-HT. We concluded that platelets may provide 5-HT crucial for the initiation of cutaneous T cell-mediated immune responses, such as CS.

A role for mast cell release of serotonin (5-HT), via Ag-specific factors derived from Thy-1+ B220+ lymphoid cells in the initiation of murine contact sensitivity (CS) has been suggested. However, because CS in mast cell-deficient mice was intact, a role for mast cells in CS initiation was unclear. Therefore, we examined whether CS could be initiated by i.v. injection of nonimmune mixed lymphoid cells that were sensitized in vitro with IgE. When naive mice received IgE-sensitized nonimmune spleen or lymph node cells, or IgE-sensitized purified mast cells, together with immune CS-effector B220- T cells, which therefore were depleted of CS-initiating, Thy-1+, B220+ cells, which could not transfer CS, then reconstitution of CS occurred. Mast cell-deficient W/Wv mice could not elicit this IgE-dependent CS ear swelling, but when mast cell deficiency was reversed by ear injection of normal bone marrow-derived cultured mast cells, then CS was restored. In vitro pretreatment with irrelevant monoclonal anti-OVA IgE prevented CS initiation mediated by Ag-specific, IgE mAb-sensitized cells, presumably by blocking sensitization with IgE. Thus Fc epsilon R on the normal lymphoid cells were involved. When ketanserin, a 5-HT2 receptor antagonist, was injected i.v. before cell transfer, CS initiation via IgE-sensitized cells and CS were no longer elicited. Thus, in this system, IgE Abs bound to circulating IgE Fc epsilon R bearing lymphoid cells sensitized in vitro (most likely basophils), probably mediated early activation of these circulating basophils to release mediators, causing 5-HT release from cutaneous mast cells, to mediate CS initiation.

Rat mast cells pretreated with the tricyclic antidepressant drug amitriptyline and stimulated with compound 48/80 secreted 60% of the total serotonin present in the cells, but only 15% of histamine, another amine stored in the same granules. Ultrastructural studies demonstrated that mast cells undergoing such differential release do not exhibit classical degranulation by compound sequential exocytosis. However, there were changes in granule shape and size, as well as alterations in many morphometric parameters consistent with secretion. Storage granules lost their homogeneity, exhibited greatly reorganized matrix and were surrounded by clear spaces which were often associated with small (0.1-0.01 microns) cytoplasmic vesicles, some of which contained electron-dense material. Secretory granules often had bud-like protrusions or were fused together in series. Quantitative autoradiography localized 3H-serotonin outside the storage granules, close to small vesicles, while staining with ruthenium red demonstrated that vesicular structures associated with differential release were not endocytotic. These results suggest that amitriptyline may inhibit regular exocytosis and permit at least serotonin to be moved selectively from storage granules to the cytosol or small vesicles from which it is eventually released.

In four different systems it was shown that murine delayed-type hypersensitivity (DTH) responses at 18-48 h were preceded by early 2-h responses. CBA mice immunized with picryl chloride, BDF1 mice immunized with oxazolone, BALB/c mice immunized with dinitrofluorobenzene, and C57BL/6 mice immunized with L5178Y lymphoma cells, and challenged with the appropriate specific antigen, all gave rise to expected 18-48 h delayed-in-time hypersensitivity reactions, but all of these responses were preceded by early hypersensitivity reactions that peaked at 2 h. These early 2-h reactions are transferable with T cells or with a T cell-derived, antigen-binding factor and are antigen-specific. The early and late components of DTH reactions are mast cell dependent since neither are elicited in mast cell deficient W/Wv or Sl/Sld mice. The T cell activity mediating the early component of DTH is demonstrable as early as 24 h after immunization, while the classical late component of DTH is not demonstrable until days 3-4. The difference in onset after immunization of the early and late components of DTH, and the different kinetics of these components in recipients of cell transfers that were challenged immediately or 24 h after transfer, led to the hypothesis that immunization for DTH leads to rapid induction in lymphoid organs of a certain population of T cells to produce an antigen-binding factor. This factor sensitizes peripheral tissues, probably mast cells, and local challenge with appropriate antigen leads to mast cell activation and release of the vasoactive amine serotonin, resulting in increased permeability of the local vasculature. This allows other circulating antigen-specific T cells, which are induced later after immunization, to enter the tissues and interact with antigen, resulting in production of chemoattractant lymphokines that recruit accessory leukocytes such as monocytes and polymorphs to enter the tissues via gaps between endothelial cells. These inflammatory cells, that are recruited to the site via two different T cell activities, constitute the characteristic infiltrate of DTH responses. Identification of an early 2-h component of DTH that is T cell- and mast cell-dependent provides evidence that the tissue-sensitizing, antigen-binding, T cell factor probably functions in vivo in the early phases of DTH responses.

Picryl chloride factor (PC1-F) is an antigen (TNP hapten)-binding T cell factor that initiates PC1 contact sensitivity (CS). PC1-F initiates PC1 CS by mediating an early 2-h skin swelling reaction that is due to local release of the vasoactive amine serotonin (5-HT) by mast cells, and perhaps other 5-HT-containing cells. Experiments were conducted to determine if PC1-F could sensitize normal mast cells in vitro for subsequent release of 3H-5-HT that had been taken up previously. It was found that PC1-F could sensitize mast cells, inasmuch as incubation with PC1-F, followed by washing, resulted in the ability to release 5-HT by challenge with Ag (TNP-bovine serum albumin), or by an anti-factor mAb called 14-30. As with release induced by anti-TNP IgE mAb PC1-F-induced release required phosphatidyl serine. Mast cell sensitization and activation for 5-HT release by PC1-F was not due to contamination of PC1-F with IgE antibody, because IgE (and not PC1-F) was sensitive to reduction and alkylation. Also, affinity columns linked with 14-30 or anti-IgE showed that the mast cell sensitizing and activating property of PC1-F was clearly separate from that of IgE. PC1-F-induced release was not IgE dependent, because mast cells that were acid-stripped and largely depleted of surface IgE, could then be sensitized by PC1-F. In vivo experiments demonstrated that local challenge with 14-30 antibody induced a 2-h ear swelling reaction in actively contact sensitized mice, or adoptive recipients of sensitized cells, and in normal mice that received PC1-F i.v. These findings suggest that in vitro sensitization of mast cells with PC1-F, and subsequent in vitro release of 5-HT induced by challenge with 14-30 antibodies, correlates with the initiation of PC1 CS in vivo. Therefore, in the initiation of CS by PC1-F, mast cells can be one source of 5-HT, to cause the early, vasoactive phase of CS.

Previous studies have shown that T cell-dependent activation of mast cells to release serotonin is required for the elicitation of delayed-type hypersensitivity in mice. We have recently described an antigen-binding T cell factor that is a suitable candidate for participation in the mechanism by which T cells activate mast cells in delayed-type hypersensitivity. The T cell factor transfers the ability to elicit an antigen-specific immediate hypersensitivity-like ear-swelling reaction following local challenge with antigen. In the current study, alterations in the morphology of local tissue mast cells and vessels were studied by light and electron microscopy at the time of optimal swelling and increase in vascular permeability. Factor-induced reactions showed mild changes in metachromatic staining of mast cell granules compared with more profound changes that were found in reactions of antigen-challenged mice that were sensitized by intravenous injection with IgE antibody. Subtle changes in the ultrastructure of mast cells in reactions induced by the T cell factor included surface activation to form filopodial extensions, resulting in a significant increase in the surface density by stereologic analysis. The cytoplasm of these mast cells also showed signs of synthetic and metabolic activity with formation of vesicles and an increased prominence of the Golgi apparatus and mitochondria. Local vessels at sites of reactions due to the T cell factor or IgE showed intravascular accumulation of polymorphonuclear leukocytes, gaps at sites of endothelial cell junctions, and occasional emigration of the leukocytes into the perivascular area. This indicates that a vasoactive factor, such as serotonin, and chemotactic factors were released in both instances. However, in recipients of the T cell factor, this was accomplished with only moderate signs of overt mast cell degranulation and loss of granule density. Instead, there was formation of vesicles at the outer margin of the granules, in the perigranular membrane, and in the cytoplasm, accompanied by the surface activation. In contrast, mast cells from reactions in IgE-sensitized animals appeared to degranulate by a process of sequential compound exocytosis with no vesicle formation or cytoplasmic findings of increased synthetic or metabolic activity. The granules of these cells showed a great loss of density and many appeared swollen, resulting in overall swelling and increase in area of the cell.(ABSTRACT TRUNCATED AT 400 WORDS)

The recent identification of a T-cell-derived antigen-binding molecule (TABM), Trichinella spiralis factor (Tric-F), isolated from culture supernatants of lymphoid cells from mice infected with the intestinal helminth T. spiralis, has led to investigation of the ability of Tric-F to induce a T-cell-dependent feedback circuit that ultimately suppresses the production of other TABMs with similar (isotype-like) features. This form of regulation that has been identified in contact hypersensitivity and in delayed-type hypersensitivity (DTH) responses to tumor cells, was shown not to be antigen-specific but to be DTH-specific. Injection of mice with the TABM called picryl chloride factor (PCl-F) induced suppression of the production of DTH-initiating TABMs of other antigenic specificities. In this study, we report that intravenous injection of mice with Tric-F or PCl-F, 8 days before an oral infection with T. spiralis, induced suppressor cells that inhibited the T-cell-dependent influx into the gut of inflammatory cells, comprising mast cells and eosinophils. Similar results were obtained when the mice were skin sensitized with PCl 8 days prior to a T. spiralis infection, i.e. in a system where TABMs are known to be produced. The phenotype of these suppressor cells was Lyt-1-2+. This suppression preferentially affected the parasite-induced DTH-like response in the gut. In contrast, increased levels of IgA plasma cells in the gut, and worm expulsion were not affected by these treatments. In reciprocal experiments, intravenous injection of Tric-F, or PCl-F, or an oral infection with T. spiralis (that results in the production of TABMs) given 8 days before contact sensitizing mice with PCl, resulted in a suppression of elicitation of cutaneous DTH, as measured by ear swelling. In contrast, pretreatment with anti-dinitrophenyl IgE antibody did not interfere with intestinal inflammation to T. spiralis nor with DTH to PCl. Our results suggest that similar to cutaneous DTH, T. spiralis-specific T-cell factors are involved in the initiation and regulation of the DTH-like mast cell and eosinophil-rich intestinal inflammation that accompanies T. spiralis infections in the gut. Since both Tric-F and PCl-F induce suppression of cellular immune responses in vivo, independent of antigen specificity, it is concluded that Tric-F belongs to the same isotype of TABMs as PCl-F that therefore can be regulated by a non-antigen-specific, isotype-like, T-cell-dependent feedback mechanism.

The growth rate of Cloudman S91 melanoma cells was compared in groups of normal and immunologically compromised DBA/2 mice that had undergone thymectomy and treatment with antilymphocyte serum. Tumor growth was markedly accelerated in the immunosuppressed animals. Other groups of normal and immunosuppressed animals were treated with daily injections of either histamine, the H-2 antihistamine cimetidine, the H-1 antihistamine pyrilamine; or the mast cell stabilizer proxicromil. Histamine treatment accelerated tumor growth, but only in normal animals and had little effect on tumor growth in immunocompromised hosts. Cimetidine treatment tended to increase tumor growth in normal hosts but this was statistically significant in only 1 of 3 experiments. In contrast, treatment with cimetidine, pyrilamine, or proxicromil always resulted in significant retardation of tumor growth in immunosuppressed animals. These data are consistent with the notion that thymectomy and treatment with antilymphocyte serum results in enhanced tumor growth that is in part due to activation of histamine-dependent suppressor cells. In this system, histamine activation of suppressor cells may be reversed by treatment with either antihistamines or proxicromil, a drug that prevents mast cell release of histamine. However, since the effects of these drugs seem to depend on the immune status of the host, thorough evaluation of immunoregulatory function and careful testing to determine whether histamine blockers reduce or promote tumor growth would seem indicated when immunomodulatory treatment with these drugs is contemplated.

The biologically active fragment of human parathormone (PTH) and intact bovine PTH were found to induce secretion of both serotonin and histamine from rat peritoneal mast cells in vitro. Release of serotonin and histamine was demonstrated with 25 units/ml PTH or higher. This level is within the higher limits of the elevated PTH levels found in advanced uremia. Mast cell secretion by PTH was dose, time and energy dependent and was not cytotoxic. Although mast cell activation was independent of extracellular calcium, it required intracellular calcium, thus resembling the action of certain other peptide secretagogues. Intradermal injection of PTH induced immediate increases in vascular permeability suggesting that PTH could induce mast cell secretion in vivo. Light and electron microscopic observations confirmed mast cell degranulation by exocytosis. These results demonstrate that elevated levels of PTH can induce mast cell secretion in vitro and in vivo and suggest a possible role for mast cells in the pathophysiology of non-allergic disease states.

The skin sites of the mouse where delayed-type hypersensitivity (DTH) reactions are most easily elicited (foot pads and ears) are particularly rich in 5-hydroxytryptamine (5-HT)-containing mast cells. Since mice are deficient in circulating basophils, which play a role in at least some DTH reactions, we investigated the possibility that the mast cells were playing an important role in the evolution of the skin reactions of DTH in mice. We found that reserpine, a drug which depletes mast cells of 5-HT, abolished the ability of the mouse to make DTH reactions in the skin. The suppressive effect of reserpine could be partially blocked by monoamine oxidase inhibitors which prevent the degradation of 5-HT in the cytosol of the mast cell. Spleen cells of immune, reserpine-treated mice transferred DTH reactions to nonimmune mice normally, indicating that the reserpine treatment did not affect immune T cells. DTH reactions could not be transferred into reserpine-treated mice. We suggest that T cells are continually emigrating from the blood, through postcapillary venule endothelium, by a mechanism which does not depend on vasoactive amines. If they are appropriately immune and meet the homologous antigen in the tissue, they induce mast cells to release vasoactive amines which cause postcapillary venule endothelial cells to separate, allowing the egress from the blood of cells which ordinarily do not recirculate. The secondarily arriving vasoactive amine-dependent cells are responsible for the micro- and macroscopic lesions of DTH reactions. Chemotactic factors may also be involved in bringing cells to the DTH reaction sites but we propose that T-cell regulation of vasoactive amine-containing cells allows the effector cells to pass through the endothelial gates after they are called.

We studied binding of serotonin to protein(s) derived from rat basophil leukemia (RBL) cells and mast cells. We found two types of serotonin binding protein in RBL cells. These proteins differed from one another in molecular weight and eluted in separate peaks from sephadex G-200 columns. Peak I protein (KD = 1.9 X 10(-6) M) was a glycoprotein that bound to concanavalin A (Con A); Peak II protein (KD1 = 4.5 X 10(-8) M; KD2 = 3.9 X 10(-6) M) did not bind to Con A. Moreover, binding of [3H]serotonin to protein of peak I was sensitive to inhibition by reserpine, while binding of [3H]serotonin to protein of peak II resisted inhibition by that drug. Other differences between the two types of binding protein were found, the most significant of which was the far more vigorous conditions of homogenization required to extract peak I than peak II protein. Neither peak I nor peak II protein resembled the serotonin binding protein (SBP) that is found in serotonergic neurons of the brain and gut. Electron microscope radioautographic analysis of the intracellular distribution of [3H]serotonin taken up in vitro by RBL cells or in vivo by murine mast cells indicated that essentially all of the labeled amine was located in cytoplasmic granules. No evidence for a pool in the cytosol was found and all granules were capable of becoming labeled. The presence of two types of intracellular serotonin binding proteins in these cells may indicate that there are two intracellular storage compartments for the amine. Both may be intragranular, but peak I protein may be associated with the granular membrane while peak II protein may be more free within the granular core. Different storage proteins may help to explain the differential release of amines from mast cell granules.

Previous studies have indicated that cutaneous mast cells are involved in the elicitation of delayed-type hypersensitivity (DTH) in mice. Mast cells are thought to be required in DTH to release serotonin to open gaps between endothelial cells, allowing entrance of effector cells into the tissue. Two different strains of mice with independent genetic defects that lead to a substantial mast cell deficiency (W/Wv and SI/SId), and their normal littermate +/+ controls, were studied for their ability to express DTH. Both strains were shown to be deficient in serotonin-containing mast cells at skin sites of preferential elicitation of DTH in normal mice, such as the ear or footpad. Defective DTH was found in both mast cell-deficient strains by using two different systems: 1) sheep erythrocyte-induced footpad DTH, and 2) picryl chloride-induced contact sensitivity ear swelling responses. Adoptive transfer experiments demonstrated that abnormal DTH in mast cell-deficient mice was due to a defect in the elicitation of DTH, rather than a defect in the induction of effector T cells. In these experiments, the ability to elicit DTH could be transferred to normal +/+ mice with sensitized cells from mast cell-deficient mice, but sensitized cells from +/+ mice could not transfer DTH responsiveness to mast cell-deficient mice. In addition, no defects in numbers of epidermal Langerhans cells or in antigen-presenting cell function were found in W/Wv or SI/SId mice. We therefore concluded that abnormal elicitation of DTH in W/Wv and SI/SId mice was probably due to their mast cell deficiency. The inability of mast cell-deficient mice to express DTH was overcome when sensitized T cells and specific antigen were placed in the extravascular tissues by local passive transfer. These results suggest that mast cell release of vasoactive mediators, such as serotonin, is required in DTH to allow effector T cells to leave the intravascular space, enter the tissues, and become activated by antigen to release chemoattractant lymphokines that recruit a nonspecific infiltrate of inflammatory cells.

It has recently been shown that the classical 24-48-h component of delayed-type hypersensitivity (DTH) in mice is preceded by an early 2-h component. This early component of DTH resembles IgE-dependent hypersensitivity reactions, but is mediated by an antigen-specific T cell factor that activates cutaneous mast cells (MC). In the current study of cutaneous hypersensitivity reactions, evidence is presented that serotonin and histamine are released from the granules of MC in IgE-dependent responses, but that T cell activation of MC induces preferential release of serotonin. In T cell-dependent reactions, it was found that serotonin was released under experimental conditions in which MC granules were depleted of serotonin, but catabolism of serotonin in the cytosol was prevented. This suggests that T cells induce differential release of serotonin from non-granule storage sites in MC, such as transport vesicles in the cytoplasm. Morphological observations of MC in IgE-dependent compared to T cell-dependent cutaneous reactions were consistent with this hypothesis. Exocytosis of granules characterized IgE activation and was not found in MC that were activated by T cells, which instead showed mild degranulation accompanied by numerous cytoplasmic vesicles.

Bone marrow-derived blood basophils are recruited into the tissues by immuno mechanisms in a variety of delayed time-course hypersensitivity responses. In the skin these are called cutaneous basophil hypersensitivity (CBH) reactions. In guinea pigs, it is now established that the elicitation of CBH is dependent on T cell- and/or (antibody)-triggered mechanisms. Both are subject to modulation. T cell-mediated CBH seems to be suppressed in basophil-poor tuberculin-type reactions. B cells mediate CBH via antibody of IgG1 isotype through mechanisms that involve Fc receptors, which can be competitively blocked. After basophils arrive at a CBH reaction they can be triggered by antigen to immediately release mediators such as histamine. Thus, one consequence of the arrival and accumulation of basophils at delayed hypersensitivity reactions is to augment the anaphylactic potential of a given tissue site. In reactions to parasites, release of mediators by tissue basophils seems to aid in the expulsion of these multicellular organisms, In addition, histamine released by recruited basophils, or by locally resident mast cells, may modulate some delayed reactions through stimulation of histamine-2 receptors on cells such as T lymphocytes. In mice, mast cell release of serotonin and subsequent stimulation of the local vasculature seems to be required to allow diapedesis and tissue accumulation of various bone marrow-derived accessory leukocytes in delayed-type hypersensitivity responses. Thus, basophils and mast cells, and their release of mediators such as vasoactive amines, are involved in the onset, development, and function of various tissue hypersensitivity responses.

We have previously suggested that the release of serotonin (5-hydroxytryptamine) (5-HT) by local tissue mast cells is required for the elicitation of delayed-type hypersensitivity (DTH) in mice. In the current study, light microscopic radioautographs from animals treated with [3H]5-HT indicated that local mast cells released 5-HT between 6 and 18 h during the evolution of DTH. Ultrastructural examination of mast cells revealed surface activation, indicated by extension of surface filopodia, and degranulation by fusion and exocytosis. Light and electron microscopic studies of the endothelium of postcapillary venules at sites of DTH revealed the development of gaps between adjacent cells. The development of gaps permitted extravasation of tracers that was abolished by depletion or antagonism of 5-HT. Thus mast cells degranulated and released 5-HT in DTH, and this 5-HT acted on local vessels. Recipients of nonadherent, non-immunoglobulin-bearing sensitized lymphocytes also demonstrated similar mast cell degranulation and the formation of endothelial gaps. This indicated that mast cell degranulation and 5-HT release in murine DTH were probably T cell dependent.

Pretreatment of mast cells with various psychotropic agents was shown to permit preferential release of serotonin without substantial release of histamine or massive degranulation. Differential release involved both endogenous, granule-stored serotonin, and exogenous radiolabeled serotonin that had been taken up by the cell. This phenomenon occurred in mast cells stimulated to secrete with suboptimal concentrations of the classic mast cell secretagogue compound 48/80, was associated with drugs of several different structures and known mechanisms of action, and could be inhibited by certain prostaglandins. Furthermore, differential release of serotonin occurred in mast cells of retired breeders without the use of drugs or other exogenous agents. Light microscopic studies of mast cells undergoing differential release showed minimal degranulation, indicating that most of the serotonin release did not occur via classic exocytosis. The ability of mast cell to selectively release serotonin, by a mechanism unlike that occurring in allergic anaphylactic secretion, constitutes one of the first instances of differential release from secretory cells, suggests a new mechanism of release of secretory products, and expands the potential role of mast cells in the pathophysiology of the body.

The involvement of mast cells in anti-tumor resistance was studied by employing 2 strains of mast cell deficient but otherwise immunocompetent mice on a C57BL/6 (H-2b) background (W/Wv and Sl/Sld) and their respective normal +/+ littermate controls. Sensitization of control mice with irradiated semisyngeneic B16 melanoma cells (H-2b) resulted in protection against subsequent challenge with viable B16 cells, in contrast to sensitization of either W/Wv or Sl/Sld mice. The involvement of serotonin in antitumor resistance was studied by employing 2 serotonin active drugs: reserpine, that depletes mast cells of serotonin; and methysergide, a serotonin antagonist. Sensitization of BDF1 mice with irradiated B16 cells and sensitization of DBA/2 mice (H-2d) with irradiated SL2 cells (H-2d) resulted in protection against subsequent challenge with viable B16 cells and viable SL2 cells, respectively. Treatment with either reserpine or methysergide resulted in a decreased protection. Delayed-type hypersensitivity (DTH) footpad responses to allogeneic L5178Y (H-2d) tumor cells in C57BL/6 mice showed a biphasic reaction pattern, similar to that found in DTH responses to simple reactive haptens, such as picryl chloride. Moreover, the early swelling responses were also dependent on T cells and on mast cells. BDF1 mice carrying a semisyngeneic L5178Y tumor on the chest showed an early swelling response after footpad challenge but no late response, possibly indicating that selective down regulation of the late component of DTH was associated with progressive tumor growth in these animals. The biphasic patterns of DTH to both tumor cells and picryl chloride and the T cell and mast cell dependence of both antitumor resistance and DTH to tumor cells suggest that T cell-dependent activation of mast cells to allow entry of mononuclear leukocytes into sites of tumor growth is similar to the mechanism that occurs in DTH.

Picryl (trinitrophenyl) chloride (PCL) contact sensitization of mice induces T cells that release an antigen-binding T cell factor (PCLF) that plays an important role in the initiation of contact sensitivity responses, in part via activation of mast cells. The current study employs an in vitro indirect rosette assay to demonstrate that PCLF can interact with the mast cell surface. Sheep red blood cells (SRBC) were hapten conjugated with trinitrophenyl (TNP), dinitrophenyl (DNP), or oxazolone (OX). When TNP-conjugated SRBC were coated with PCLF, monoclonal anti-DNP IgE, or anti-DNP IgG1, they produced 40 to 50% rosettes with purified normal mouse peritoneal mast cells. Analogous antigen-binding factors, from lymphoid cells of OX and dinitrofluorobenzene contact-sensitized mice, gave similar mast cell rosetting levels with OX-SRBC and DNP-SRBC, respectively. PCLF demonstrated a high degree of hapten specificity in that it formed rosettes with TNP-SRBC but not with DNP-SRBC, unlike IgE and IgG1, or DNPF, which formed rosettes with either SRBC type. Similarly, soluble TNP-BSA could inhibit PCLF rosette-forming capacity, but soluble DNP-BSA could not. In addition to mouse mast cells, PCLF formed rosettes with rat basophil leukemia cells, mouse peritoneal exudate macrophages, mouse alveolar macrophages, and J 774 cultured mouse macrophages; it did not form rosettes with rat mast cells, rat alveolar macrophages, or mouse spleen cells. Thus, PCLF-formed rosettes were antigen specific, relatively species specific, and mast cell/macrophage specific. PCLF-mediated rosette-forming activity could be detected in the presence of nanogram quantities of PCLF. More than 10 times greater IgE was needed to produce IgE-mediated rosettes. Reduction and alkylation eliminated the rosetting activity of IgE, but the rosetting activity of PCLF was not affected. PCLF, but not IgE rosette-forming activity, could be removed by and eluted from affinity columns linked with a monoclonal antibody specific for T cell-derived antigen-binding factors, whereas PCLF rosetting activity was not retained by an anti-immunoglobulin affinity column. Preincubation of mast cells with rat myeloma IgE or mouse monoclonal IgE of various specificities blocked IgE rosettes but not PCLF-induced rosettes. Other immunoglobulin isotypes likewise did not block PCLF rosettes. However, PCLF rosettes could be blocked by preincubation of mast cells with OX factor (OXF),and OXF-mediated rosettes could be blocked similarly by PCLF. These results suggest that the antigen-binding T cell factor PCLF interacts with a unique receptor on the surface of mouse mast cells.

Atopic dermatitis (AD) usually develops in patients with an individual or family history of allergic diseases, and is characterized by chronic relapsing inflammation seen especially in childhood, association with IgE hyperproduction and precipitation by environmental factors. However, the exact etiology of AD has been unclear. To further explore the pathogenesis and treatment of AD, a suitable animal model is required. We found that skin lesions, which were clinically and histologically very similar to human AD, spontaneously appeared on the face, neck, ears and dorsal skin of inbred NC/Nga mice when they were raised in non-sterile (conventional) circumstances, but not under specific pathogen-free conditions. Plasma levels of total IgE in conventional NC/Nga mice were markedly elevated from 8 weeks of age, correlating with clinical skin severity of dermatitis. Immunohistochemical examination of the skin lesion showed increased numbers of mast cells and CD4+ T cells containing IL-4 necessary for IgE synthesis. Thus, NC/Nga mice suffered from dermatitis very similar to human AD with IgE hyperproduction, which may be triggered by some environmental factor(s). [ABSTRACT FROM AUTHOR]