Detecting antibodies with similar reactivity patterns in the HLDA8 blind panel of flow cytometry data.

The blind panel collected for the 8th Human Leucocyte Differentiation Antigens Workshop (HLDA8; ) included 49 antibodies of known CD specificities and 76 antibodies of unknown specificity. We have identified groups of antibodies showing similar patterns of reactivity that need to be investigated by biochemical methods to evaluate whether the antibodies within these groups are reacting with the same molecule. Our approach to data analysis was based on the work of Salganik et al. (in press) [Salganik, M.P., Milford E.L., Hardie D.L., Shaw, S., Wand, M.P., in press. Classifying antibodies using flow cytometry data: class prediction and class discovery. Biometrical Journal].

A study of titanium release into body organs following the insertion of single threaded screw implants into the mandibles of sheep.

BACKGROUND: Titanium is generally considered a safe metal to use in implantation but some studies have suggested that particulate titanium may cause health problems either at the site overlying the implant or in distant organs, particularly after frictional wear of a medical prosthesis. It was the purpose of this investigation to study the levels of dissemination of titanium from threaded screw type implants following placement of single implants in sheep mandibles. METHOD: Twelve sheep were implanted with a single 10x3.75mm self-tapping implant for time intervals of one, four and eight to 12 weeks. Four unoperated sheep served as controls. Regional lymph nodes, lungs, spleens and livers were dissected, frozen and subsequently analysed by Graphite Furnace Atomic Absorption Spectroscopy. RESULTS: Results associated with successful implants showed no statistically significant different levels of titanium in any organ compared to controls, although some minor elevations in titanium levels within the lungs and regional lymph nodes were noted. Two implants failed to integrate and these showed higher levels of titanium in the lungs (2.2-3.8 times the mean of the controls) and regional lymph nodes (7-9.4 times the levels in controls). CONCLUSIONS: Debris from a single implant insertion is at such a low level that it is unlikely to pose a health problem. Even though the number of failed implants was low, multiple failed implants may result in considerably more titanium release which can track through the regional lymph nodes. Results suggest that sheep would be an excellent model for following biological changes associated with successful and failed implants and the effect this may have on titanium release.

Migration of Langerhans cells and gammadelta dendritic cells from UV-B-irradiated sheep skin.

Depletion of dendritic cells from UV-B-irradiated sheep skin was investigated by monitoring migration of these cells towards regional lymph nodes. By creating and cannulating pseudoafferent lymphatic vessels draining a defined region of skin, migrating cells were collected and enumerated throughout the response to UV-B irradiation. In the present study, the effects of exposing sheep flank skin to UV-B radiation clearly demonstrated a dose-dependent increase in the migration of Langerhans cells (LC) from the UV-B-exposed area to the draining lymph node. The range of UV-B doses assessed in this study included 2.7 kJ/m2, a suberythemal dose; 8 kJ/m2, 1 minimal erythemal dose (MED); 20.1 kJ/m2; 40.2 kJ/m2; and 80.4 kJ/m2, 10 MED. The LC were the cells most sensitive to UV-B treatment, with exposure to 8 kJ/m2 or greater reproducibly causing a significant increase in migration. Migration of gammadelta+ dendritic cells (gammadelta+ DC) from irradiated skin was also triggered by exposure to UV-B radiation, but dose dependency was not evident within the range of UV-B doses examined. This, in conjunction with the lack of any consistent correlation between either the timing or magnitude of migration peaks of these two cell types, suggests that different mechanisms govern the egress of LC and gammadelta+ DC from the skin. It is concluded that the depression of normal immune function in the skin after exposure to erythemal doses of UV-B radiation is associated with changes in the migration patterns of epidermal dendritic cells to local lymph nodes.

Ultraviolet light induced injury: immunological and inflammatory effects.

This article reviews many of the complex events that occur after cutaneous ultraviolet (UV) exposure. The inflammatory changes of acute exposure of the skin include erythema (sunburn), the production of inflammatory mediators, alteration of vascular responses and an inflammatory cell infiltrate. Damage to proteins and DNA accumulates within skin cells and characteristic morphological changes occur in keratinocytes and other skin cells. When a cell becomes damaged irreparably by UV exposure, cell death follows via apoptotic mechanisms. Alterations in cutaneous and systemic immunity occur as a result of the UV-induced inflammation and damage, including changes in the production of cytokines by keratinocytes and other skin-associated cells, alteration of adhesion molecule expression and the loss of APC function within the skin. These changes lead to the generation of suppressor T cells, the induction of antigen-specific immunosuppression and a lowering of cell-mediated immunity. These events impair the immune system's capacity to reject highly antigenic skin cancers. This review gives an overview of the acute inflammatory and immunological events associated with cutaneous UV exposure, which are important to consider before dealing with the complex interactions that occur with chronic UV exposure, leading to photocarcinogenesis.

UV-induced changes in the skin: can they be repaired?

The damaging effects of UVB light have been described previously and include a number of changes to multiple cell types. At previous meetings of this society, we have shown that Langerhans' cells are the most susceptible to UVB induced damage which can be shown as ultrastructural changes in dendrites, nucleus and cytoplasm by transmission electron microscopy. We have also shown that their patterns of migration from skin to regional lymph node and their ability to present antigens to autologous T cells have been profoundly altered by UVB irradiation. The aim of this work was to establish if it was possible to reverse any of the damage done to Langerhans' cells by UVB exposure by topical application of a DNA repair enzyme such as T4N5 endonuclease. These experiments were undertaken in a sheep model that allowed collection of cells as they migrate from the skin. This allowed for a direct examination of the migration characteristics and ultrastructural features of all Langerhans' cells before, during, and for 2 weeks after exposure to a single dose of UVB. Results obtained from this project indicate that treatment by topical application of DNA repair enzyme immediately after UVB irradiation may restore a number of normal immune parameters associated with the structure and function of migrating Langerhans' cells. It appears that there is a dose related correction of the increased tempo of cell migration and some improvements in the number of ultrastructurally damaged Langerhans' cells have also been associated with application of higher doses of DNA repair enzyme. These preliminary findings indicate that some potential therapeutic benefits are associated with the use of such agents in reversing the immunological damage caused by exposure to erythemal doses of UVB light.

Effects of UV on the migration and function of epidermal antigen presenting cells.

Depletion of epidermal Langerhans cells (LC) and the concomitant depression of the skin immune system after excessive exposure to ultraviolet B light (UVB) has been established in the international literature for some time. Our investigations were intended to determine whether or not these phenomena occurred as a direct result of increased LC migration being triggered by the UVB exposure. To test this hypothesis, a sheep model was established in which the lymphatic vessels draining a defined region of skin were cannulated and the cells migrating towards the regional lymph node continuously collected. Cell populations in these collections were identified and enumerated by indirect immunofluorescence and flow cytometry. These experiments showed there was a significant, dose-dependent increase in the rate of LC migration from sheep skin after exposure to doses of UVB light exceeding 1 minimal erythemal dose (MED). In a series of parallel experiments, the functional characteristics of dendritic cells (DC) migrating from normal or UVB irradiated sheep were studied. To assay them, enriched preparations of DC were collected via cannulated afferent lymphatic vessels and pulsed with antigen prior to incubation with autologous peripheral blood lymphocytes. The relative efficiency of antigen presentation was determined by the ability of DC to induce T cell proliferation. Our data clearly demonstrate that there is a profound loss of normal antigen-presenting cell function after exposure to UVB light. Various experiments were undertaken to determine the mechanism(s) associated with these changes in migration kinetics and cellular function. Electron microscopic examinations of LC migrating from normal or UVB irradiated skin have demonstrated a profound loss of dendritic processes after UVB exposure. This provides a possible explanation for the changes in skin immunity after UVB exposure.

Down-regulation of an established immune response via chemical carcinogen or UVB-altered skin.

The ability to produce antigen-specific down-regulation of an established immune response was investigated in 2,4,6-trinitrochlorobenzene (TNCB)-immune mice by delivery of antigen through chemical carcinogen- or ultraviolet B (UVB)-treated skin. When TNCB-immune mice were treated on the dorsal trunk skin with 7,12-dimethylbenz(a)anthracene (DMBA) followed by TNCB there was an antigen-specific reduction in both contact sensitivity and antibody production. Further, immune mice that received spleen cells from naive syngeneic donors treated with DMBA followed by TNCB also exhibited a reduction in both contact sensitivity and antibody production. In contrast, mice treated with UVB irradiation followed by TNCB had a reduction in contact sensitivity but not antibody production. These results provide evidence that an ongoing immune response can be manipulated by immunization through a modified skin immune system. This may provide a beneficial approach for the treatment of autoimmune disease.

Failure of carcinogen-altered dendritic cells to initiate T cell proliferation is associated with reduced IL-1 beta secretion.

The activation of T cells through presentation of antigen by dendritic cells (DC) relies on many factors, including the correct balance of cytokines in the immediate microenvironment. Antigen presentation by DC migrating from carcinogen-treated skin is impaired as evidenced by the failure of antigen-pulsed DC to initiate specific T cell proliferation. To elucidate mechanism(s) of DC dysfunction, DC migrating from carcinogen-treated skin were collected, pulsed with OVA, and cultured with antigen-specific autologous lymphocytes. Supernatants were assayed for the costimulatory cytokine IL-1 beta which influences the outcome of DC:T cell interactions. The dendritic cells migrating from carcinogen-treated skin that failed to induce T cell proliferation were unable to produce IL-1 beta. This may account for the abrogation of DC function following exposure to chemical carcinogens and provides an explanation for the inability of DC to induce a protective immune response to carcinogen-induced tumours.

Abrogation of afferent lymph dendritic cell function after cutaneously applied chemical carcinogens.

Chemical carcinogens reduce cutaneous immunity, an event accompanied by alterations to the number and morphology of the resident epidermal Langerhans cell (LC) population. This study aimed to examine the functional capacity of LC and other dendritic cells (DC) that are migrating from carcinogen-treated skin via afferent lymphatic vessels. Generation and subsequent cannulation of prefemoral pseudoafferent lymphatic vessels in sheep allowed continuous collection of DC migrating from a defined area of carcinogen-treated skin. The ability of metrizamide-enriched afferent lymph DC to present antigen to autologous primed peripheral blood lymphocytes was used as an indicator of DC function. Topical application of the complete carcinogens 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene abrogated the stimulatory capacity of migrating DC for periods of 8 weeks and 5 weeks, respectively, whereas the tumor promoter 12-O-tetradecanoylphorbol-13-acetate reduced DC function for less than 1 week. These findings favor tumor development in carcinogen-treated skin being enhanced due to impairment of DC immunological surveillance.

The migration of Langerhans' cells into and out of lymph nodes draining normal, carcinogen and antigen-treated sheep skin.

Epidermal Langerhans' cell (LC) migration to the regional lymph node and beyond into central lymph was examined in sheep following topical application of the complete chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) or the contact sensitizing antigen 2,4,6-trinitrochlorobenzene (TNCB). This was facilitated by cannulating previously constructed pseudoafferent lymphatic vessels draining the skin treated with these agents or alternatively, the efferent lymphatic vessel of the regional lymph node. Application of DMBA resulted in a biphasic increase in LC migration. There was an initial increase in LC migration at 25 h with the maximum response (3.6 x 10(7) LC/h) occurring approximately 5 days after DMBA treatment. In contrast, the contact sensitizing antigen TNCB caused enhanced LC migration within minutes of the application of antigen (3.3 x 10(6) LC/h) and peak migration at 8-12 h. Examination of efferent lymph cells from the regional lymph node after DMBA treatment showed uncharacteristically large numbers of LC traversing the lymph node. These LC migration patterns suggest different mechanisms may trigger the migration of LC from skin after the application of DMBA to those associated with the normal processes of antigen presentation.

Langerhans cell migration patterns from sheep skin following topical application of carcinogens.

Application of tumour promoters or complete chemical carcinogens to skin alters the density and/or morphology of epidermal Langerhans cells (LC). To examine the hypothesis that these chemical carcinogens alter LC migration kinetics from the epidermis, pseudoafferent lymphatic vessels draining defined areas of carcinogen treated sheep skin were cannulated and the number of LC migrating enumerated using indirect immunofluorescence and flow cytometry. The complete carcinogen benzo[a]pyrene (BP) and the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) caused an immediate two to four-fold increase in the rate of LC migration, while the tumour initiator urethane did not alter LC migration. The antigenicity of the carcinogens utilized was assessed in contact hypersensitivity experiments in mice. BP and TPA were mildly antigenic whereas urethane failed to initiate a contact hypersensitivity response in sensitized mice. It is concluded that the initial increase in LC migration from skin following the application of the tumour promoter TPA and the complete carcinogen BP is partly due to LC recognizing these carcinogens as antigens.

Langerhans cell alterations in cutaneous carcinogenesis.

Analysis of the early stages of experimental skin cancer in mice has demonstrated that complete chemical carcinogens (e.g. DMBA or benzo(a)pyrene) and tumour promoters (e.g. TPA) but not tumour initiators (e.g. urethane) deplete or functionally alter epidermal Langerhans cells (LC). These changes result in altered local immunity as antigen presentation through LC depleted skin results in either immune tolerance due to the generation of suppressor T cells or anergy. Parallel studies in sheep have shown that, following the application of DMBA, depletion of LC is due to increased migration of these cells from the skin whereas tumour initiators did not alter LC migration. Likewise benzo(a)pyrene did not trigger enhanced LC migration from the epidermis. Experiments in mice suggest that part of this increased migration after the application of DMBA is due to the carcinogen being handled as an antigen by the epidermal LC. However, this fails to explain the prolonged migration which follows. The implication of these studies is that early in carcinogenesis, altered immune function occurs as a result of LC depletion/modification, allowing aberrant cells to proliferate in the absence of immune destruction.

Migration of Langerhans cells from carcinogen-treated sheep skin.

To define the mechanism(s) of carcinogen depletion of Langerhans cells (LC) from skin, the migration of LC from the skin to the regional lymph node was examined in carcinogen-treated, antigen-treated, and control sheep. This was assessed by cannulation of afferent lymphatic vessels that drain the treated areas of skin or the efferent lymphatic draining the regional lymph node. Cells draining from test or control skin were continuously collected and enumerated by indirect immunofluorescence and flow cytometry using specific anti-CD1 monoclonal antibodies. There was a marked increase in the rate of LC migration in the 8 h following the application of the contact sensitizing antigen trinitrochlorobenzene (TNCB). The chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) triggered a tenfold-greater migration of LC compared with TNCB--with the peak response at 5 d. After DMBA treatment LC were also detected in the efferent lymph of the regional lymph node. It is concluded that the depletion of LC from carcinogen-treated skin is due to the increased LC migration and not carcinogen-induced cell death.

The effect of substance P on the regional lymph node antibody response to antigenic stimulation in capsaicin-pretreated rats.

The undecapeptide substance P (SP) contained in primary afferent nerves is thought to mediate that part of the neurogenic inflammatory response consisting of vasodilation and plasma extravasation. This response is diminished in rats pretreated as neonates with the neurotoxin capsaicin. It is not known whether primary afferent nerves influence cellular responses of the immune response to antigenic stimulation. Using 6- to 12-wk-old Sprague-Dawley rats pretreated as neonates with capsaicin, we examined the regional lymph node response to a s.c. antigenic stimulus of sheep red blood cells. The number of cells secreting antigen-specific antibody in these animals was reduced by more than 80% using direct and indirect plaque assay methods. The reduced antibody response in capsaicin-pretreated animals was reversed by a s.c. infusion of SP given over a 4-hr period at the injection site immediately after antigen stimulation. This response had a threshold at approximately 1.0 X 10(-5) M SP. SP1-7 (1.0 X 10(-5) M) was without effect but an infusion of SP5-11 (1.0 X 10(-5) M) reversed the effects of capsaicin treatment indicating a carboxyl-terminal effect of SP. The results suggest that the reduced response of capsaicin-treated animals to an antigenic stimulus is due to an effect of capsaicin on the SP-containing primary afferent nerves rather than a toxic effect of capsaicin on the immune system.

The migration of lymphocytes into rat popliteal lymph nodes during antigenic promotion or competition between heterologous erythrocytes.

The injection of chicken and sheep red blood cells (CRBC and SRBC) into rat popliteal lymph nodes either together or sequentially 2, 4, 6, or 8 days apart resulted in an enhanced immune response when the second antigen was injected 2 or 4 days after the injection of the first antigen (antigenic promotion) or a suppressed immune response when the second antigen was injected 6 days after the injection of the first antigen (antigenic competition). The immune response to either antigen was dependent upon the time of administration of the second antigen with respect to the first antigen. Lymphocyte migration into antigenically stimulated lymph nodes was greater when the two antigens were injected sequentially rather than together. Further, the migration of lymphocytes into the lymph node was enhanced when the second antigen was injected during the inductive or suppressive phase of the immune response to the first antigen (CRBC) regardless of whether the same (CRBC) or an antigenically unrelated antigen (SRBC) was used as the second antigen. While antigenic promotion may in part be explained by the increased rate at which lymphocytes migrate into lymph nodes, lymphocyte migration is also enhanced during antigenic competition. This suggests that while suppressor cells/factors may regulate the effector phase of an immune response they do not directly modulate the migration of blood-borne lymphocytes into the lymph node.

The migration of blood-borne lymphocytes into rat popliteal lymph nodes stimulated with xenogeneic red blood cells.

The recruitment of radiolabelled blood-borne lymphocytes into rat popliteal lymph nodes (PLN) was investigated following the injection of varying doses of chicken (CRBC) or sheep red blood cells (SRBC) into the hind foot pad. Within the dose range tested (10(5)-10(8) RBC) an increase in the dose of injected antigen resulted in elevated levels of lymphocyte recruitment into the draining popliteal lymph node. The kinetics of lymphocyte recruitment with the same dose of CRBC or SRBC was similar even though these red cells differ in size and are antigenically non-cross-reactive. While little is known of the mechanisms which control the rate of entry of blood-borne lymphocytes into antigen-stimulated lymph nodes, the extent of lymphocyte recruitment was shown to be directly related to the quantity of antigen injected.