Stimulatory lipids accumulate in the mouse liver within 30 min of contact sensitization to facilitate the activation of Naïve iNKT cells in a CD1d-dependent fashion.

Natural killer T cells with invariant αß-T cell receptors (TCRs) (iNKT cells) constitute a lipid-responsive arm of the innate immune system that has been implicated in the regulation or promotion of various immune, infectious and neoplastic processes. Contact sensitivity (CS), also known as contact hypersensitivity or allergic contact dermatitis, is one such immune process that begins with topical sensitization to an allergen and culminates in a localized cutaneous inflammatory response after challenge with the same allergen. CS depends on events initiated early in sensitization by hepatic iNKT cells. We have shown previously that these iNKT cells release IL-4 early after skin sensitization to activate B-1 B cells to produce IgM antibodies that aid in local recruitment of the effector T cells. Here, we utilize adoptive transfer techniques in several strains of knockout mice to demonstrate that hepatic lipids isolated 30 min after sensitization are significantly more stimulatory to naïve hepatic iNKT cells than hepatic lipids isolated after sham sensitization. These stimulatory hepatic lipids specifically affect iNKT cells and not B-1 B cells. The downstream CS response is abrogated with anti-CD1d-blocking antibodies, suggesting a critical role of CD1d in the activation of hepatic iNKT cells with these lipids. Hepatocytes may not be essential, as donor hepatic iNKT cells can reconstitute CS without migrating to the recipient mouse liver. Rather, CD1d-expressing liver mononuclear cells are sufficient for activation of iNKT cells. In conclusion, stimulatory lipids accumulate in the liver soon after sensitization and facilitate iNKT cell activation in a CD1d-dependent yet potentially hepatocyte-independent manner.

Participation of iNKT cells in the early and late components of Tc1-mediated DNFB contact sensitivity: cooperative role of γδ-T cells.

Prior studies of classical 24 h responses in TNP-Cl (picryl chloride) allergic contact sensitivity (CS), showed mediation by Th1 cells in CBA mice, and established that 24 h elicitation of responses requires an early 2 h CS-initiating component dependent on iNKT cells, IL-4 and B-1 B cells. Here, we studied the other form of cytotoxic T cell (Tc1) CS in DNFB sensitized BALB/c mice and determined that similar CS-initiation also is required. We systematically tested each step of the initiation pathway in this model. Thus, DNFB Tc1 CS was significantly impaired in iNKT cell deficient CD1d(-/-) and Jα18(-/-) mice, IL4Rα(-/-) and STAT-6(-/-) mice, and also in pan B-cell deficient JH(-/-) mice. Further, the Tc1 DNFB CS-initiating component, like Th1 response to TNP-Cl, was elicited by only 1-day after immunization, due to B-1 cells. In summary, we show that CS-Initiation also is required in Tc1 CS. Further, we have newly determined regulatory support of both the early and late components of DNFB induced Tc1 CS by iNKT cells and γδ-T cells. In summary, both iNKT cells and assisting γδ-T cells are involved in initiating and effector phases of DNFB induced CS.

Valvular heart disease in patients with hypocomplementemic urticarial vasculitis syndrome associated with Jaccoud's arthropathy.

BACKGROUND: Since 1973, more than 75 patients with hypocomplementemic urticarial vasculitis syndrome (HUVS) were reported, but valvular heart disease does not seem to have been noted in these patients. Since 1993, however, five patients with HUVS accompanied by Jaccoud's arthropathy (JA) were found to have serious valvular heart disease. METHODS: To characterize the cardiac valvulopathy of the third patient with HUVS/JA to have undergone valve replacement, this study included the use of routine and special tissue stains, as well as immunohistochemical staining. We compared gross and histologic findings of this patient's valve to those of two other patients with this complex syndrome who underwent valve replacement. Pathologic findings of these latter two patients were described in separate earlier reports. RESULTS: Histologic examination of the resected valves in all three patients showed an acute necrotizing endocarditis and fibrin deposition on the surface of valve leaflets. Beneath the surfaces of the leaflets, there was evidence of chronic inflammation, consisting of lymphocytes and histiocytes. A fibrocalcific degenerative change was also present in all three valves. Positive staining for IgG, IgA, IgM, and light-chain determinant-bearing proteins was detected primarily at the valve surface in special studies of the aortic valve of the patient described in the current report. CONCLUSION: Patients with HUVS and associated JA should be evaluated for the presence of valvular heart disease. The latter is probably a nonrheumatic, inflammatory, and degenerative process, mediated by immune complex, as well as cellular immune mechanisms.

Topical tacrolimus and cyclosporin A differentially inhibit early and late effector phases of cutaneous delayed-type and immunoglobulin E hypersensitivity.

Systemic and topical administration routes of tacrolimus and cyclosporin A (CsA) were compared in effects on early and late phases of elicited T-cell-mediated contact sensitivity (CS), and effects on early and late phases of cutaneous immunoglobulin E (IgE) antibody-mediated hypersensitivity responses in mice. Thus, both CS and IgE responses in the skin have an early mast-cell-dependent phase, and also a late inflammatory phase. We measured the effects of both immunosuppressants on both phases of the respective T cell versus IgE responses. Systemic administration of both agents completely suppressed CS and IgE late-phase responses, but failed to affect either early phase. In contrast, when topical CsA was used, low doses abolished the early phase of IgE responses, but even high doses did not inhibit the early phase of CS. Conversely, topical tacrolimus inhibited the early phase of CS more potently than the early phase of cutaneous IgE hypersensitivity responses. Thus, topical treatment was needed to inhibit the early phases and the two agents acted differentially, suggesting differing susceptibility of the early phases, that are probably due to different signalling mechanisms. These studies underscore the potential value of topical administration of these powerful immunosuppressive agents in the treatment of allergic diseases that exhibit features of early-phase mast-cell-dependent inflammation, and late inflammation due to mast cells or to T cells, such as atopic dermatitis or asthma, since the early phase is predominantly susceptible to topical application, while the last phase of both IgE and T-cell inflammation responds to systemic treatment with both agents.

Yes T cells, but three different T cells (alphabeta, gammadelta and NK T cells), and also B-1 cells mediate contact sensitivity.

Transfer of contact sensitivity (CS) responses by immune lymphoid cells was the first finding that distinguished cellular from humoral immunity. CS has remained the most studied T cell reaction in vivo, and is the prototype for a variety of delayed-type hypersensitivity (DTH) responses. DTH in essence is the recruitment of effector alphabeta-T cells out of vessels into peripheral tissues. The T cells then are activated by antigen presenting cells to produce pro-inflammatory cytokines. It has been assumed that the alphabeta-T cells alone are responsible, but recent studies show that three other lymphocyte subsets are involved: CS-inducing NK T cells, CS-initiating B-1 cells, and CS-assisting gammadelta-T cells. Therefore, the effector alphabeta-T cells are essential, but cannot be recruited into the tissues without the local action of IgM antibodies produced by B-1 cells rapidly (1 day) post-immunization. The IgM complexes with the challenge antigen to locally activate complement to lead to vascular activation required for T cell recruitment. This process occurs early (1-2 hours) in the elicitation phase, and is called CS-initiation. The essential CS-inducing NK T cells activate the B-1 cells by producing IL-4 rapidly (1 hour) after immunization, and gammadelta-T cells assist the local inflammatory function of the recruited CS-effector alphabeta-T cells. Thus, four lymphocyte subsets are required for elicitation of responses: CS-inducing NK T cells, CS-initiating B-1 cells, CS-assisting gammadelta-T cells, and finally the CS-effector alphabeta-T cells. Three of these four cell types are present in the immune lymphoid cell population that adoptively transfers CS: B-1 cells, gammadelta-T cells, and the alphabeta-T cells.

T cytotoxic 1 and T cytotoxic 2 CD8 T cells both inhibit IgE responses.

BACKGROUND: It is well recognized that CD8 T cells inhibit IgE responses. In this study, we investigated the mechanism of CD8 T cell-mediated IgE suppression by comparing the capacity of T cytotoxic 1 (Tc1) and T cytotoxic 2 (Tc2) CD8 T cells to inhibit IgE responses to ovalbumin (OVA). METHODS: Tc1 and Tc2 CD8 T cells were generated from OVA(257-264)-specific Vbeta5.2 T cell receptor (TcR) transgenic mice by stimulation with anti-CD3 and anti-CD28 under Tc1 and Tc2 polarizing conditions. Tc1 and Tc2 Vbeta5.2 TcR CD8 T cells (10(6)) were adoptively transferred to syngeneic mice, and following immunization with 100 micro of OVA/alum, serum IgE antibodies were measured by passive cutaneous anaphylaxis and expressed as the highest dilution that gave a detectable skin response. RESULTS: Both Tc1 and Tc2 CD8 T cells from OT-I mice inhibited IgE. CONCLUSION: Both Tc1 and Tc2 CD8 T cells promote Th1 immunity and inhibit IgE responses. This process appears to be independent of CD8 T cell-derived IFN-gamma, as both Tc2 (IFN-gamma-) and Tc1 (IFN-gamma+) CD8 T cells inhibited IgE.

Allergen-specific Th1 cells counteract efferent Th2 cell-dependent bronchial hyperresponsiveness and eosinophilic inflammation partly via IFN-gamma.

Th2 T cell immune-driven inflammation plays an important role in allergic asthma. We studied the effect of counterbalancing Th1 T cells in an asthma model in Brown Norway rats that favors Th2 responses. Rats received i.v. transfers of syngeneic allergen-specific Th1 or Th2 cells, 24 h before aerosol exposure to allergen, and were studied 18-24 h later. Adoptive transfer of OVA-specific Th2 cells, but not Th1 cells, and OVA, but not BSA exposure, induced bronchial hyperresponsiveness (BHR) to acetylcholine and eosinophilia in a cell number-dependent manner. Importantly, cotransfer of OVA-specific Th1 cells dose-dependently reversed BHR and bronchoalveolar lavage (BAL) eosinophilia, but not mucosal eosinophilia. OVA-specific Th1 cells transferred alone induced mucosal eosinophilia, but neither BHR nor BAL eosinophilia. Th1 suppression of BHR and BAL eosinophilia was allergen specific, since cotransfer of BSA-specific Th1 cells with the OVA-specific Th2 cells was not inhibitory when OVA aerosol alone was used, but was suppressive with OVA and BSA challenge. Furthermore, recipients of Th1 cells alone had increased gene expression for IFN-gamma in the lungs, while those receiving Th2 cells alone showed increased IL-4 mRNA. Importantly, induction of these Th2 cytokines was inhibited in recipients of combined Th1 and Th2 cells. Anti-IFN-gamma treatment attenuated the down-regulatory effect of Th1 cells. Allergen-specific Th1 cells down-regulate efferent Th2 cytokine-dependent BHR and BAL eosinophilia in an asthma model via mechanisms that depend on IFN-gamma. Therapy designed to control the efferent phase of established asthma by augmenting down-regulatory Th1 counterbalancing mechanisms should be effective.

Basophil responses to chemokines are regulated by both sequential and cooperative receptor signaling.

To investigate human basophil responses to chemokines, we have developed a sensitive assay that uses flow cytometry to measure leukocyte shape change as a marker of cell responsiveness. PBMC were isolated from the blood of volunteers. Basophils were identified as a single population of cells that stained positive for IL-3Ralpha (CDw123) and negative for HLA-DR, and their increase in forward scatter (as a result of cell shape change) in response to chemokines was measured. Shape change responses of basophils to chemokines were highly reproducible, with a rank order of potency: monocyte chemoattractant protein (MCP) 4 (peak at <1 nM) >/= eotaxin-2 = eotaxin-3 >/= eotaxin > MCP-1 = MCP-3 > macrophage-inflammatory protein-1alpha > RANTES = MCP-2 = IL-8. The CCR4-selective ligand macrophage-derived chemokine did not elicit a response at concentrations up to 10 nM. Blocking mAbs to CCR2 and CCR3 demonstrated that responses to higher concentrations (>10 nM) of MCP-1 were mediated by CCR3 rather than CCR2, whereas MCP-4 exhibited a biphasic response consistent with sequential activation of CCR3 at lower concentrations and CCR2 at 10 nM MCP-4 and above. In contrast, responses to MCP-3 were blocked only in the presence of both mAbs, but not after pretreatment with either anti-CCR2 or anti-CCR3 mAb alone. These patterns of receptor usage were different from those seen for eosinophils and monocytes. We suggest that cooperation between CCRs might be a mechanism for preferential recruitment of basophils, as occurs in tissue hypersensitivity responses in vivo.

B-1 B cell IgM antibody initiates T cell elicitation of contact sensitivity.

Although B-1 B cells have received considerable attention, their actual role in the normal functioning of the immune system is unclear. The hypothesized role of B-1 cell IgM in natural protective immunity is just being established. We have uncovered a separate and novel role for B-1 cell IgM in initiating the elicitation of acquired T cell-dependent contact sensitivity (CS), the prototype of in vivo T cell immunity, early after immunization (within 4 days). The recent recognition of a similarly unanticipated role of B cells in a variety of T cell responses, may indicate that B-1 cell IgM has a broader role in immunity than thought previously. We showed that 24 hr CS responses, and rises in local IFN-gamma levels at 24 hrs later after antigen (Ag) challenge the ears, were absent in pan B cell and antibody deficient mice. The mechanism of B cell involvement in CS-initiation is via local C5a generation early (1-2 hrs) after antigen (Ag) challenge of the ears, in 4 day contact sensitized mice. C5a activates local mast cells to release serotonin (5-HT) and TNF alpha to induce endothelial ICAM-1 and VCAM-1, leading to T cell recruitment. We hypothesized that C5a was generated via complement activation due to antibodies forming local AgAb complexes, and that B-1 cell IgM was involved because isotype switching of B-2 cells to produce C-activating IgG isotypes, could not occur as early as day 4. Indeed, B-1 cell deficient CBA/N-xid mice lacked C5a in 2 hr ear extracts, and had impaired CS ear swelling and elaboration of IFN-gamma at 24 hrs. Importantly, adoptive transfer of purified normal peritoneal B-1 cells, or just i.v. injection of Ag-specific IgM monoclonal antibodies in sensitized xid, restored deficient early C5a and late 24 hr ear swelling. These results suggest that early after Ag challenge, specific B-1 cell IgM, produced at distant sites by prior sensitization, forms AgAb complexes that trigger elaboration of C5a, to activate mast cell release of vasoactive TNF alpha and 5-HT to initiate CS, leading to T cell recruitment. We postulate that antibody of various isotypes possibly may lead to local vascular activation to aid in T cell recruitment in a variety of T cell responses, but that very early after immunization, Ag-specific IgM produced by B-1 cells, preferentially serves this important function.

IL-12 reverses established tolerance mediated by TCRalphabeta+ but not by TCRgammadelta+ suppressor T cells.

Topical cutaneous painting with chemically reactive haptens induces the ability to subsequently elicit contact sensitivity (CS) responses in the skin. These CS responses are in vivo examples of acquired, antigen (Ag)-specific T cell immunity, and are a form of delayed-type hypersensitivity (DTH). In contrast, high dose i.v. administration of the hapten can induce Ag-specific tolerance. In some instances this specific immune hyporeactivity is due to suppressor T cells. We investigated the effect of IL-12 on reversal of tolerance due to suppressor T cells that were induced by i.v. administration of hapten in either normal TCRalpha+/+, or in immunodeficient TCRalpha-/- mice. In the TCRalpha+/+ mice, tolerance is mediated by TCRalphabeta+ suppressor T cells, while in the TCRalpha-/- mice the tolerance is due to suppressive TCRgammadelta+ cells. Treatment with IL-12 reversed suppressor mediated by the TCRalphabeta+ cells, but did not affect tolerance due to TCRgammadelta+ suppressor cells. Another difference was that the alphabetaTCR+ suppressor cells produced a soluble suppressor factor that could replace the surppressor cells, while gammadeltaTCR+ suppressor cells did not. We hypothesized that IL-12 may strengthen responses of target CS-effector T cells influenced by the hapten-MHC-specificity of alphabeta suppresssor cells, or suppressor factor. On the other hand, gammadeltaTCR+ suppressive cells likely have specificity for the hapten alone, and are not MHC-restricted, and therefore probably do not operate via peptide-MHC interactions, that could be strengthened by IL-12. The ability of IL-12 to strengthen the resistance of CS-effector T cells to alphabeta TCR suppressor cells, may be due to the ability of IL-12 to increase T cell costimulation mediated by signaling mechanisms acting via B7.1 and B7.2. In contrast, gammadeltaTCR+ suppressor cells, that are largely hapten-specific, probably do not interact with peptide/MHC complexes on APC, and thus are not affected by IL-12 strengthening of co-stimulation.

Early local generation of C5a initiates the elicitation of contact sensitivity by leading to early T cell recruitment.

We have shown previously that an early complement C5-dependent cascade is required to recruit T cells to elicit 24-h contact sensitivity (CS) responses. In this paper, we have characterized molecular events of this early required cascade by biochemically analyzing extracts of mouse ears undergoing elicitation of CS. Chemotactic activity was found after local Ag challenge, in CS ear extracts early (by 1 h), in CS ear extracts late (through 24 h), in previously immunized mice, but not in ears of vehicle-immunized or non-immune-challenged mice. The early chemotactic activity at 2 h was likely caused by C5a, because it was neutralized in vitro by anti-C5a Ab, was inactive on C5aR-deficient (C5aR-/-) macrophages, and was absent in C5-deficient mice. The activity was present in T cell-deficient mice, but elaboration was Ag-specific. This T cell-independent, Ag-specific elaboration of C5a early in CS ear responses likely led to T cell recruitment, because subsequent local IFN-gamma mRNA and protein expression, as markers of T cell arrival and activation, began by 4 h after Ag challenge. In contrast to early C5a chemotactic activity, late chemotactic activity 24 h after Ag challenge was unaffected by anti-C5, was active on C5aR-/- macrophages, was T cell-dependent, and by ELISA appeared largely due to chemokines (macrophage-inflammatory protein-1alpha and -1beta, IFN-gamma-inducible protein-10, and monocyte chemoattractant protein-1). Importantly, early generation of C5a was required for T cell recruitment because C5aR-/- mice had absent 24-h CS. Taken together, these findings indicate an important linkage of C5a as a component of early activated innate immunity that is required for later elicitation of acquired T cell immunity, probably by facilitating the initial recruitment of T cells into the Ag-challenged local site in CS responses.

IL-12 is produced by antigen-presenting cells stimulated with soluble alphabeta TCR and restores impaired T(h)1 responses.

Contact sensitivity (CS) is a cutaneous T(h)1 response that is induced by skin painting with reactive hapten. In prior in vivo studies of CS, we showed that recombinant soluble alphabetaTCR (sTCR) acted non-specifically to protect CS-effector T cells from suppression, but no molecular mechanism was determined. In the current study, we employed an in vitro system to investigate the mechanism of how sTCR protect CS-effector T cells from suppression. Immune CS-effector cells and appropriate hapten-conjugated antigen-presenting cells (APC) were incubated together with down-regulatory culture supernatant produced by suppressive spleen cells from mice tolerized i.v. with specific hapten, which produced strong inhibition of IFN-gamma production by the CS-effector cells. Importantly, addition of two different sTCR, of unrelated specificity, reversed this down-regulation and thus restored IFN-gamma production. We found that the APC, and not the CS-effector T cells, were the locus of the sTCR-mediated protection and showed direct binding of sTCR to APC by flow cytometry. Further, addition of anti-IL-12 showed that sTCR protection was due to IL-12 induced by sTCR and released by the APC, and was confirmed by ELISA measurement of IL-12 induced in APC supernatants by sTCR incubation. These results indicated a possible new regulatory loop in which suppression was reversed by IL-12 derived from APC, following direct surface binding of sTCR, and enhanced by IFN-gamma production from the T(h)1 CS-effector cells.

Endothelial cell E- and P-selectin up-regulation in murine contact sensitivity is prolonged by distinct mechanisms occurring in sequence.

The selectins are adhesion molecules that mediate the tethering and rolling of leukocytes on vascular endothelium. Although E-selectin and P-selectin are known to be expressed by endothelial cells (EC) in response to proinflammatory stimuli, their pattern and mechanisms of expression in immune-mediated inflammation remain poorly understood. By quantifying luminal endothelial selectin expression via i.v. administration of radiolabeled mAb, we detected constitutive expression of P-selectin, but not E-selectin, in mouse skin. Both selectins were transiently up-regulated after intradermal TNF-alpha, IL-1alpha, or IL-1beta. In contrast, during a contact sensitivity response to oxazolone, expression of both selectins was prolonged, with distinct peaks at 6 and 48 h. Experiments with P-selectin gene-targeted mice showed that the P-selectin measured was exclusively expressed by EC rather than platelets. The early and late phases of selectin expression in contact sensitivity were differentiated in terms of their requirement for prior sensitization, and the action of IL-1. Whereas the early phase was a nonspecific 'irritant' response to oxazolone, the late phase was Ag specific and was partially IL-1 dependent. Therefore, persistence of both E- and P-selectin expression in vivo can occur as a result of sequential and distinct EC activation processes that appear to be at least partially different from those previously reported as stimulating ICAM-1 and VCAM-1 expression. The further elucidation of mechanisms of EC activation in this model may help determine the relative roles of selectins and ligands for leukocyte integrins in the sequential recruitment of T cells and other leukocyte subsets during ongoing immune-mediated inflammatory responses.

C-C chemokines in allergen-induced late-phase cutaneous responses in atopic subjects: association of eotaxin with early 6-hour eosinophils, and of eotaxin-2 and monocyte chemoattractant protein-4 with the later 24-hour tissue eosinophilia, and relationship to basophils and other C-C chemokines (monocyte chemoattractant protein-3 and RANTES).

The relationship of expression of the C-C chemokines eotaxin, eotaxin 2, RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4 to the kinetics of infiltrating eosinophils, basophils, and other inflammatory cells was examined in allergen-induced, late-phase allergic reactions in the skin of human atopic subjects. EG2+ eosinophils peaked at 6 h and correlated significantly with eotaxin mRNA and protein, whereas declining eosinophils at 24 h correlated significantly with eotaxin-2 and MCP-4 mRNA. In contrast, no significant correlations were observed between BB1+ basophil infiltrates, which peaked at 24 h, and expression of eotaxin, eotaxin-2, RANTES, MCP-3, and MCP-4 or elastase+ neutrophils (6-h peak), CD3+ and CD4+ T cells (24 h), and CD68+ macrophages (72 h). Furthermore, 83% of eosinophils, 40% of basophils, and 1% of CD3+ cells expressed the eotaxin receptor CCR3, while eotaxin protein was expressed by 43% of macrophages, 81% of endothelial cells, and 6% of T cells (6%). These data suggest that 1) eotaxin has a role in the early 6-h recruitment of eosinophils, while eotaxin-2 and MCP-4 appear to be involved in later 24-h infiltration of these CCR3+ cells; 2) different mechanisms may guide the early vs late eosinophilia; and 3) other chemokines and receptors may be involved in basophil accumulation of allergic tissue reactions in human skin.

Role of interleukin-4 in down-regulation of contact sensitivity by gammadelta T cells from tolerized T-cell receptor alpha-/- mice.

Contact sensitivity (CS) is a classical example of an in vivo T-cell-mediated immune response that is under regulation. Such down-regulation can be mediated by alphabeta T cells in mice that are tolerized by prior exposure to high doses of antigen. In contrast, we demonstrated previously that such high-dose antigen tolerance in T-cell receptor (TCR) alpha-/- H-2d mice induced antigen-specific, apparently major histocompatibility complex-unrestricted, CD4- CD8- gammadelta T cells, that also could down-regulate CS responses antigen-specifically in vivo, and also inhibited in vitro production of IFN-gamma. In the present experiments we employed H-2b-deficient TCRalpha-/- and TCRbeta-/- mice, owing to different molecular constructs than were used previously, and confirmed that tolerized gammadelta T cells in these different H-2b alphabeta TCR-/- mice down-regulated CS. Thus, gammadelta T-cell suppressor function was not limited to mice bearing a special transgenic TCRalpha-/- DNA construct. Furthermore, employing monoclonal antibody and complement depletion in vitro and adoptive transfer in vivo, characterized the phenotype of these gammadelta down-regulatory T cells as: CD3+, CD28+, CD40-ligand+, Fas+, FcgammaR+ and NK1.1-. Also, in vitro antigen desensitization of these trinitrophenyl (TNP)-specific TCRgammadelta+ down-regulatory cells was achieved with soluble TNP-bovine serum albumin (BSA), but not with oxazolone-BSA, showing that these suppressive gammadelta T cells have antigen-specific receptors. Moreover, employing monoclonal antibody blocking of gammadelta suppressors in vitro, and of recipients in vivo, we showed that interleukin-4 (IL-4) was involved in this down-regulation of CS by gammadelta T cells, while IL-10 and transforming growth factor-beta2 were not. In summary, generation of antigen-specific, double-negative, gammadelta suppressor cells, by tolerance of high antigen doses in TCRalpha-/- mice, appears to be a general phenomenon, and IL-4 production is involved in their down-regulation of the T helper type 1 cells that mediate CS.

A new paradigm of T cell allergy: requirement for the B-1 cell subset.

BACKGROUND: We have uncovered a role for B-1-B-cell-produced IgM antibody, in the initiation of contact sensitivity (CS) in mice. CS and delayed-type hypersensitivity (DTH) involve recruitment of T cells to the tissues, to be activated by antigen-presenting cells (APC), and then make cytokines. Little is known about low recruitment is initiated. In CS, soon after immunization, the unique B-1 cell subset, responsible for the formation of most IgM, is activated to produce antigen (Ag)-specific IgM for export to tissues. IgM forms complexes with challenge Ag, activating the classical complement (C) pathway, generating C5a, to activate endothelium directly, or indirectly via C5a receptors (R) on mast cells and platelets, that release vasoactive amines (serotonin) and cytokines (TNF-alpha). These act together to induce vasodilatation, vascular permeability and expression of endothelial adhesion molecules to promote optimal T cell recruitment. METHODS AND RESULTS: New findings that established this pathway include: (1) absent CS response in C-deficient, or C-inhibited mice; (2) local generation of C5a in CS tissue extracts; (3) absent CS in C5aR-/- mice; (4) decreased CS in B cell and B-1-cell-deficient mice, and (5) reconstitution of CS by transfer of B-1 cells, or hapten-specific IgM. CONCLUSION: These findings indicate that the B-1 subset producing Ag-specific IgM is required early in CS to activate C, to induce vasoactive mediators that initiate local T recruitment.

Human allogeneic vascular rejection after arterial transplantation and peripheral lymphoid reconstitution in severe combined immunodeficient mice.

BACKGROUND: Interspecies differences create important shortcomings in existing animal models used to describe in vivo events responsible for allograft rejection. Alloimmune destruction of human dermal microvessels, histologically consistent with rejection, has been demonstrated in human skin-grafted severe combined immunodeficient (SCID) mice receiving allogeneic human peripheral blood mononuclear cells (PBMC). We have now documented human alloimmune injury in a vascularized, SCID-human arterial transplantation model. METHODS: Fresh human artery was used to replace the CB.17 SCID/beige mouse infrarenal aorta. Seven days later, 3x10(8) human PBMC were administered intraperitoneally, and lymphocyte engraftment was considered successful when circulating human CD3+ cells were later identified in peripheral blood. RESULTS: Forty-six of 49 (94%) mice undergoing transplantation survived, including 14 controls with arterial grafts receiving no PBMC. Twenty-eight of 32 mice demonstrated circulating human CD3+ cells, 14 days after PBMC administration. Animals were killed at 14, 21, or 28 days after receiving allogeneic PBMC, and arteries were recovered for histology and immunohistology. All 14 control mice had patent transplanted grafts with normal vascular histology and no lymphoid infiltration. Damage to transplanted arteries among lymphocyte-engrafted mice was apparent by 14 and 21 days in some animals, whereas 16 of 22 exhibited moderate to severe intimal, medial, and/or adventitial lymphocytic infiltration with intimal expansion by day 28. The infiltrate consisted of HLA-A, -B, -C+, and -DR+, human CD3+ cells, approximately equally distributed as CD4+ and CD8+ subsets. Some infiltrating lymphocytes were cytolytic cells as demonstrated by perforin staining. The endothelium of transplanted human arteries exhibited endothelialitis, and the endothelial cells stained intensely with anti-HLA-A, -B, -C and anti-HLA-DR antibodies. The expanded intima was predominantly smooth muscle cells, staining positively for smooth muscle alpha-actin, HLA-A, -B, -C and HLA-DR. Medial necrosis was not observed. CONCLUSION: The results provide evidence of alloimmune-mediated vascular rejection in this human arterial transplantation model.