Passive transfer of experimental hypersensitivity pneumonitis with lymphoid cells in the rabbit

Although precipitating antibody is associated with human hypersensitivity pneumonitis, there is evidence that cell-mediated hypersensitivity may be involved in disease pathogenesis. In this study, interstitial, and peribronchial lesions were produced by respiratory challenge of rabbits passively sensitized with ovalbumin-sensitive lymph node cells. Ovalbumin sensitivity of donor rabbits and lymph node cells was demonstrated by skin testing, migration inhibition factor (MIF) production using alveolar wash cells as migrating cells, and lymphocyte stimulation. Passive cell transfer was accomplished by intraperitoneal injection with all lymph node cells obtained from one donor transferred to one recipient. Recipients were challenged by aerosol or intratracheal injection of antigen immediately or 24 hr after passive sensitization and were killed 48 hr after challenge. Lesions in rabbits passively sensitized by lymph node cells and challenged with antigen by intratracheal inoculation consisted of focal pneumonitis with intra-alveolar edema and infiltrates of mononuclear cells in alveoli and alveoli septa. Aerosol challenge of passively sensitized animals produced similar changes, but peribronchial tissue containing macrophages and germinal centers was prominent in this group. Antiovalbumin serum recipients challenged by intratracheal injection demonstrated only mild peribronchial mononuclear cell infiltrates, without pneumonitis. Control animals receiving lymph node cells only or challenge only demonstrated no changes in lung histology.     

A terminal-labelling microcytotoxicity assay with 125I-iododeoxyuridine as a label for target cells

The development of a terminal-labelling microcytotoxicity assay is described in which target cells (fetal fibroblasts) were labelled with 125I-iododeoxyuridine after effector (lymphoid) cells had been incubated with them for 24 h. The time-course for the development of cell-mediated cytotoxicity was assessed following allogeneic skin grafting. 'Non-specific' cytotoxicity detracts from the sensitivity of all microcytotoxicity assays and the terminal-labelling assay using 125I is no exception. The non-specific effects can be reduced but not eliminated by the removal of adherent cells. The optimum target cell/effector cell ratio would seem to be between 1:100 and 1:250. Residual lymph node cells did not appear to incorporate enough label to affect the test results. In vivo correlates of in vitro findings are still not easy to determine.     

Antigen acquisition by dendritic cells: intestinal dendritic cells acquire antigen administered orally and can prime naive T cells in vivo

In the rat, mesenteric lymphadenectomy allows collection of dendritic cells (DC) derived from the small intestine after cannulation of the thoracic duct. We prepared rats this way and administered antigens by oral feeding or intraintestinal injection. DC enriched from the thoracic duct lymph collected over the first 24 h from these animals are able to stimulate sensitized T cells in vitro and to prime popliteal lymph node CD4+ T cells after footpad injection, while B and T cells from the same thoracic duct lymph are inert in priming. 500 or less DC pulsed in vitro with antigen can prime T cells in vivo, whereas 100 times more B cells or macrophages pulsed in vitro are quite inert. 1 mg of ovalbumin administered orally is sufficient to load DC for in vivo priming of T cells. Antigen could not be detected directly in DC but was present in macrophages in the lamina propria. Direct presentation of antigen by DC to T cells was demonstrated by injecting F1 recipients with parental DC and showing restriction of T cell sensitization to the major histocompatibility complex of the injected DC. Antigen-bearing DC do not induce a detectable primary antibody response but a small secondary antibody response can be detected after a boosting injection. These results show that acquisition of antigens by DC in the intestine is very similar to what occurs in vitro or in other tissues, suggesting that there may be no special difference in antigen handling at mucosal surfaces. One implication of these results is that hypotheses designed to explain oral tolerance must take into account the presence of immunostimulatory, antigen-bearing DC in animals that have received oral antigens.     

Susceptibility to effects of UVB irradiation on induction of contact sensitivity, relevance of number and function of Langerhans cells and epidermal macrophages

Sensitization on skin exposed to acute low-dose UVB irradiation separates normal humans into two phenotypically distinct groups: One group, following sensitization on UVB-irradiated skin, develops contact sensitivity, designated UVB resistant (UVB-R) and the second group, following sensitization on UVB-irradiated skin, fails to develop contact sensitivity, designated UVB susceptible (UVB-S). To investigate whether UVB susceptibility in humans in related to antigen-presenting activity in the skin we studied the effect of UVB irradiation on the number and function of the epidermal antigen-presenting cells in volunteers identified as UVB-R and UVB-S. Single cell suspensions of epidermal cells from control skin and skin exposed to 3 minimal erythema doses (MED) of UVB 3 days previously were stained for Langerhans cells (CD1a+HLA-DR+) and epidermal macrophages (CD1a-HLA-DR+). The UVB exposure of the skin significantly decreased the percentage of Langerhans cells (UVB-R: n = 7, P < 0.02, UVB-S: n = 6, P < 0.03) and increased the percentage of epidermal macrophages (UVB-R: n = 7, P < 0.03, UVB-S: n = 6, P < 0.03) however to the same degree in both the UVB-R and the UVB-S group. To study the effect on Langerhans cell alloreactivity, epidermal cells were harvested immediately after UVB irradiation. However, in both UVB-R and UVB-S subjects the Langerhans cell alloreactivity was blocked to the same degree immediately after UVB irradiation compared to nonirradiated epidermal cells. To determine the effect of UVB irradiation on epidermal macrophages, epidermal cells were harvested 3 days after UVB irradiation. Irradiated epidermal cells from both UVB-R and UVB-S subjects demonstrated a strong antigen-presenting capacity compared to epidermal cells from control skin leading to activation of T cells that mainly secrete interferon (IFN)-gamma and not interleukin (IL)-4. In conclusion we found that UVB susceptibility was not correlated with the number of Langerhans cells or epidermal macrophages in the skin at the same time of sensitization. Neither was it correlated with the capacity of Langerhans cells nor UVB-induced epidermal macrophages to activate T cells in vitro.     

Contribution of dermal macrophage trafficking in the sensitization phase of contact hypersensitivity

We investigated cellular trafficking of dermal macrophages that express a macrophage calcium-type lectin (MMGL) during the sensitization of delayed-type hypersensitivity. In skin, dermal macrophages, but not epidermal Langerhans cells, have been shown to express MMGL. Epicutaneous sensitization by FITC produced a transient increase in MMGL-positive cells in regional lymph nodes. To directly investigate whether the increase was due to cell migration from dermis, MMGL-positive cells purified from skin were intradermally injected into syngeneic mice after labeling with a fluorescent cell tracer, followed by epicutaneous sensitization over the site of injection. MMGL-positive cells containing the tracer were found in the regional lymph nodes after sensitization. The majority of the MMGL-positive cell migrants were negative for FITC fluorescence despite the presence of FITC-labeled cells that included Langerhans cell migrants. Because the extent of MMGL-positive cell migration was greatly influenced by the selection of vehicles to dissolve FITC, the efficiency of sensitization was compared using the ear swelling test. Migration of both Langerhans cells (FITC-labeled cells) and MMGL-positive cells contributed positively to the efficiency of sensitization. Interestingly, MMGL-positive cell migration was induced by vehicle alone, even in the absence of FITC. These results suggest that migration of dermal MMGL-positive cells accounts for the adjuvant effects of vehicles at least in part.     

The role of chemokines and accessory cells in the immunoregulation of cutaneous leishmaniasis

The course of infection with Leishmania parasites is determined by the type of the developing CD4+ T cell immune response. Macrophages and Langerhans cells/dendritic cells play a decisive role in the interaction between the parasites and the host's immune system because they serve as host cells, as accessory cells that present parasite antigen, deliver costimulatory signals and secrete cytokines modulating the T cell activity and as effector cells eliminating the microorganisms. Therefore, we put particular emphasis on characterizing the role of these cells in cutaneous leishmaniasis and the factors regulating their activities. Our results show that (1) expression of the chemokine monocyte chemoattractant protein 1 (MCP-1) is associated with macrophage infiltration into the lesion and stimulation of leishmanicidal activity, (2) Langerhans cells are required for the transport of Leishmania from the infected skin to the draining lymph node and initiation of the specific T cell immune response in the early phase of infection, (3) lymph node dendritic cells containing persistent parasites may be involved in the maintenance of specific immunity, (4) Langerhans cells are able to present L. major LPG to T cells and (5) treatment of mice with antigen-pulsed Langerhans cells induces protective immunity against cutaneous leishmaniasis.     

Expression of a retrovirally transduced gene under control of an internal housekeeping gene promoter does not persist due to methylation and is restored partially by 5-azacytidine treatment

BACKGROUND: Tachykinins, such as substance P, might be involved in the development of airway hyperresponsiveness and airway inflammation. OBJECTIVE: This study was designed to investigate the effects of the tachykinin NK1 receptor antagonist SR 140333 (Nolpitantium) and the NK2 receptor antagonist SR 48968 (Saredutant) on the activation of alveolar macrophages in the guinea-pig. METHODS: Guinea-pigs sensitized and challenged by ovalbumin administered by aerosol or naive guinea-pigs were exposed by aerosol to the neutral endopeptidase, phosphoramidon and, 15 min later, to substance P. Twenty-four hours later, bronchoalveolar lavages were performed and the cell composition of bronchoalveolar lavage fluids and the arachidonate release from alveolar macrophages stimulated in vitro with fMLP were evaluated. RESULTS: Antigen challenge in sensitized guinea-pigs induced an increase in the total number of cells and granulocytes in the bronchoalveolar lavage fluids that was not reduced by pre-treatment of guinea-pigs with a single dose of SR 140333 or SR 48968 (1 mg/kg). Substance P exposure in phosphoramidon-pretreated guinea-pigs did not induce an increase in the total number of cells. In contrast, antigen or substance P exposure induced a significant increase in the in vitro fMLP-induced arachidonate release from alveolar macrophages. Pre-treatment of the guinea pigs with SR 140333 or SR 48968 did not reduce the increase in arachidonate release from fMLP-stimulated alveolar macrophages from sensitized and challenged guinea-pigs. Pre-treatment of the animals by SR 140333 and SR 48968 reduced the enhanced arachidonate release induced by fMLP from substance P-exposed guinea-pigs. CONCLUSION: The present data demonstrate the importance of NK1- and NK2-receptor stimulation in the development of substance P-induced increased reactivity of alveolar macrophages.     

Oral tolerance is determined at the level of draining lymph nodes

In the skin and in the epithelium of the oral mucosa a comparable network of Langerhans cells can be found. Antigen application on either epithelium leads to rapid emigration of Langerhans cells to the draining lymph nodes. Application on the oral mucosa leads to tolerance induction while application on the skin results in sensitization of the animal. Here we show that there are no differences in the antigen presentation capacity of oral mucosa- and skin-derived dendritic cells. However, measurement of IFN-gamma and IL-5 production, as representatives of Th1 and Th2 cytokines respectively, in total lymph node suspensions after sensitization via the skin or oral mucosa demonstrated a skewing of the response towards Th2 after antigen application on the oral mucosa. Together with our previous studies, in which it was shown that oral tolerance induction is not inherent to oral mucosa-derived dendritic cells, we postulate that oral tolerance is determined at the level of draining lymph nodes influenced by local cytokine profiles.     

ATP-induced, P2U purinoceptor-mediated constriction of isolated, perfused mesenteric beds of the rat

Upon antigen encounter epidermal Langerhans cells (LC) and dendritic cells (DC) emigrate from peripheral organs and invade lymph nodes through the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Part of this process is mimicked by metastasizing tumor cells. Since splice variants of CD44 promote metastasis to lymph nodes we explored the expression of CD44 proteins on migrating LC and DC. We show that following antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6 and v9. Antibodies against CD44 epitopes arrest LC in the epidermis, prevent the binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.     

Synergistic effects of mineral fibres and cigarette smoke on the production of tumour necrosis factor by alveolar macrophages of rats

The objective of this study was to evaluate the combined effects of mineral fibres and cigarette smoke on the production of tumour necrosis factor (TNF) by alveolar macrophages. Rats were exposed to cigarette smoke in vivo, and production of TNF by alveolar macrophages was measured in the presence of mineral fibres in vitro. For smoke exposure, rats were divided into two groups. Five were exposed to a daily concentration of 10 mg/m3 of cigarette smoke for an eight hour period, and five rats (controls) were not exposed to smoke. Bronchoalveolar lavage was performed after exposure to smoke and the recovered alveolar macrophages were incubated with either chrysotile or ceramic fibres on a microplate for 24 hours. Activity of TNF in the supernatant was determined by the L-929 fibroblast cell bioassay. When alveolar macrophages were not stimulated by mineral fibres, production of TNF by rats exposed to smoke and unexposed rats was essentially the same. When alveolar macrophages were stimulated in vitro by chrysotile or ceramic fibres, production of TNF by alveolar macrophages from rats exposed to smoke was higher than that by alveolar macrophages from unexposed rats. The findings suggest that cigarette smoke and mineral fibres have a synergistic effect on TNF production by alveolar macrophages.     

Modulation of Langerhans cell phenotype, migration and maturation by agents known to cause herpes simplex virus reactivation in a mouse model

The factors which control whether an asymptomatic or symptomatic infection in the skin follows reactivation of herpes simplex virus (HSV) in the sensory ganglia remain unclear. Xylene, retinoic acid and dimethylsuphoxide (DMSO) all stimulate similar levels of virus reactivation in the ganglia of latently infected mice, yet give rise to high, moderate and very low incidences of clinical skin disease, respectively. This observation suggests that the chemicals may be capable of affecting the local microenvironment of the skin. In the present study we have investigated the effects of xylene, retinoic acid and DMSO on Langerhans cell (LC) phenotype and function. The results show that none of the chemicals inhibit the phenotypic maturation of Langerhans cells in vitro. However, DMSO induced a dramatic elevation in class II MHC expression on Langerhans cells. In addition, fewer antigen-bearing dendritic cells (DC) were evident in the lymph nodes if xylene was administered to the skin prior to challenge with the contact sensitizer, FITC. None of the chemicals inhibited the accumulation of DC in the lymph nodes after such an antigen-challenge, suggesting that xylene was affecting not migration, but antigen uptake. The inhibition of antigen uptake by xylene together with the activation of LC in terms of class II expression by DMSO may explain, in part, the relative abilities of these chemicals to allow the establishment of recurrent HSV disease in the skin.     

Localization of tumor-reactive lymph node lymphocytes in vivo using radiolabeled monoclonal antibody

BACKGROUND: Lymph node lymphocytes vary in their responsiveness to tumor. A technique has been developed that uses radiolabeled monoclonal antibody (MoAb) against the tumor-associated mucin, TAG-72, and a gamma-detecting probe by which lymph nodes containing microscopic tumor and/or shed TAG-72 can be identified in vivo. The immunologic characteristics of these lymph nodes were examined. METHODS: Patients with colon cancer received 125I-labeled MoAb CC49 by intravenous injection preoperatively. During laparotomy lymph nodes that appeared normal on inspection and palpation but which contained radiolabeled MoAb were identified using a hand-held gamma-detecting probe. These lymph nodes and other lymph node and tumor specimens were resected for analysis. RESULTS: Lymph nodes identified by the probe were found by immunohistochemical studies to contain microscopic tumor and/or shed antigen associated with germinal centers. They were characterized by greater CD4+:CD8+ ratios, rates of expansion, and cytolytic activity compared with lymphocytes from lymph nodes with macroscopic tumor, noninvolved lymph nodes, and tumors. All lymph node lymphocytes identified by the probe demonstrated significant proliferative responses to autologous tumor and, in contrast to lymphocytes from noninvolved lymph nodes, significant proliferative responses to allogeneic TAG-72+ tumor cells and to soluble TAG-72+ mucin. CONCLUSIONS: By locating lymph nodes with microscopic tumor and/or shed antigen, the use of radiolabeled MoAb in vivo can be used to reproducibly identify tumor-reactive lymph node lymphocytes. This technique may be useful in identifying cells for use in adoptive immunotherapy programs and in studying the regulation of immune responses in vivo.     

Sturcture and function of the mononuclear phagocytic system (MPS) in chronic rhinosinusitis. A light and electron microscopic investigation (author's transl)

Neither the concept of the Reticulo-Endothelial-System (RES) Aschoff's (1924) nor that of the Reticulo-Histiocyte-System (RHS) provides a satisfactory framework into which the present knowledge of the phagocytic mononuclear cells can be fitted. Current knowledge concerning morphology, histochemistry (peroxydase and esterase activity), immunology (specific surface antigens, receptors on the cell membranes), function (immune phagocytosis, pinocytosis), kinetics (3H-thymidine labelling) and culture makes it possible to place all highly phagocytic mononuclear cells and their precursors in one system, which is called the Mononuclear-Phagocytic-System (MPS) (Langevoort, Cohn, Hirsch, Humphrey, Spector, van Furth, 1969). Kinetic studies with labelled cells have shown, that mononuclear phagocytes originate from precursor cells in the bone marrow (stem cell leads to monoblasts leads to promonocytes), than are circulating in the peripheral blood as monocytes and are transformed to tissue macrophages entering tissues. The MPS comprises following cells in following organs: connective tissue (histiocytes resp. macrophages); liver (Kupffer-cells); lung (alveolar macrophages); lymph nodes (free and fixed macrophages); bone marrow (macrophages); serous cavities (pleural and peritoneal macrophages); bone tissue (osteoclasts?); nervous system (microglial cells) (SEE Table 1). The reticular cells, endothelial cells and fibroblasts (fibrocytes) can therefore not be included in the MPS. Besides differences in morphology, histochemistry and function, they derive from mesenchymal cells and not from the bone marrow as the MPS. The present investigation demonstrates the structure and significance of the MPS in various kinds of chronic-specific and non-specific rhinosinusitis. On semithin sections two kinds of macrophages can be distinguished light-microscopically: 1. Larger macrophages with many phagosomes (storage cells) (Fig. 1A), which can exhibit sometimes a ring-shape on sections embracing greater parts of the interstitium (Fig. 1B). Such forms are mainly found in chronic (maxillary) sinusitis and are interpretated as "scavenger" macrophages. 2. The second type consists of smaller macrophages with extremely ruffling of the cell surface, which is interpretated as an expression of highly (specific?) stimulated states. These later macrophages can be seen mainly in edematous nasal polyps, which might be caused by allergic reactions of the anaphylactic type. The fine structure of the phagocytes is to some extent dependent on the actual development and functional state: there are "immature" macrophages, which are practically indistinguishable from blood monocytes (Fig. 2A); some of them can be stimulated and can therefore show many surface foldings and projections (Fig. 2B). The "mature" macrophage shows a well developed Golgi-area and many secondary lysosomes (Fig. 3). The storage type of the macrophages, which can predominate in some cases of chronic maxillary sinusitis, is characterized by many electron-lucent vacuoles (Fig. 4)...     

Reduction in number and morphologic alterations of Langerhans cells after UVB radiation in vivo are accompanied by an influx of monocytoid cells into the epidermis

Acute, low-dose ultraviolet B (UVB) radiation impairs contact hypersensitivity induction in mice by a mechanism due at least in part to Langerhans cells alterations. To better define the effects of UVB on Langerhans cells, we have compared the action of this agent on the skin of intact mice and in skin explants incubated in vitro up to 24 h. Using immunofluorescence, we detected a reduction in the length of the dendrites of Langerhans cells and a significant reduction in the number of Ia-positive Langerhans cells per unit area within 2 h of UVB; these changes reversed within 24 h in vivo, but not in vitro. By electron microscopy, the number of dendritic cells per 100 basal keratinocytes increased in vivo, but decreased in vitro by 2 h after UVB, a discordance that was significant. On the contrary, the number of dendrite profiles per dendritic cell body decreased significantly 2 h after UVB, both in vivo and in vitro. Many epidermal dendritic cells, 2 h after UVB in vivo, were deficient in cytoplasmic organelles, whereas the few cells that remained after UVB in vitro retained their Birbeck granules, and displayed many, dilated cytoplasmic vesicles. We interpret these data to mean that low doses of UVB radiation destroy the functional and morphologic integrity of epidermal Langerhans cells, and that these cells are rapidly replaced by precursor cells that mature in situ into normal-appearing Langerhans cells.     

Binding of Candida albicans yeast cells to mouse popliteal lymph node tissue is mediated by macrophages

We previously reported that Candida albicans yeast cells adhere to the macrophage-rich medullary and subcapsular sinus areas of mouse lymph node tissue. To determine whether the yeast cell-lymph node interaction is mediated by macrophages, the effect of specific elimination of macrophages on yeast cell binding was studied, and yeast cell adherence was correlated with the ingestion of India ink by lymph node cells. Macrophage elimination was done by use of liposome-containing dichloromethylene diphosphonate (L-Cl2MDP). Mice were injected in the hind footpads with the L-Cl2MDP preparation, popliteal lymph nodes were removed 5 days later, and yeast cell adherence was determined by an ex vivo binding assay. As controls, lymph nodes from mice that received footpad injections of either phosphate-buffered saline (PBS) alone or liposome-containing PBS were used. Use of macrophage- and neutrophil-specific monoclonal antibodies in tissue immunostaining showed that the L-Cl2MDP treatment eliminated macrophages but not neutrophils from the medullary and subcapsular sinus areas of the popliteal lymph nodes. A striking reduction of yeast cell adherence occurred with lymph nodes from L-Cl2MDP-treated mice compared with lymph nodes from control animals. The lymph node-yeast cell binding patterns of L-Cl2MDP-treated and control mice were the same regardless of mouse strain, sex, or T-cell competency. Results of India ink experiments, in which India ink was injected into footpads of mice and was rapidly taken up by popliteal lymph node macrophages, showed a strong correlation between yeast adherence and India ink staining of cells. In addition, the interaction of yeast cells with lymph node tissue from normal mice was not significantly affected by the addition of two extracellular matrix proteins, fibronectin and laminin, during the ex vivo adherence assay. These data indicate that medullary and subcapsular sinus lymph node macrophages express an adhesion system similar to that described for mouse splenic marginal zone macrophages.     

Neutrophils, differentiated macrophages, and monocyte/macrophage antigen presenting cells infiltrate murine epidermis after UV injury

We asked whether, as in humans, a population of antigen-presenting macrophages infiltrates the epidermis of ultraviolet (UV)-exposed BALB/c mice. Using three-color flow cytometry on cell suspensions plus in situ immunofluorescence microscopy, the phenotype of normal Langerhans cells was class II major histocompatibility complex (MHC+), CD11b+, NLDC-145+, BM8+ CD45+ and homogeneous. By contrast, in epidermal cells harvested 3 d following UV (UV-EC), there were two subsets of class II MHC+ cells: 1) class II MHChi CD11b+, and 2) class II MHClo CD11b-. Neither expressed the Langerhans cell markers BM8 and NLDC-145. In addition, there were two major populations of class II MHC- CD11b+ cells; half of these expressed the GR-1 neutrophil marker. Langerhans and dendritic epidermal T cells were markedly reduced after UV injury. By electron microscopy, immunomagnetic bead-purified CD11b+ cells in UV-EC were comprised of neutrophils, differentiated macrophages, and mononuclear cells with prominent lysosomes, but no Birbeck granules; the class II MHC+ subset resembled a monocytic cell in between differentiated macrophages and indeterminate dendritic cells. Functionally, immediately following in vivo UV exposure, the allogeneic antigen-presenting cell capacity of UV-EC was reduced to 21 +/- 6% of control epidermal cells (C-EC); by 3 d, antigen-presenting cell activity of UV-EC had recovered to 59 +/- 11% of C-EC, although at this time NLDC-145+ Langerhans cells had reached their lowest number. The recovered antigen-presenting cell activity was critically dependent upon the class II MHChiCD11b+ cells. Sensitization of BALB/c mice through skin that contained these antigen-presenting cells (3 d after UV) resulted in tolerance to dinitrofluorobenzene. By contrast, sensitization through UV-exposed skin immediately after the exposure resulted in unresponsiveness without tolerance, demonstrating temporal association of tolerance with leukocytic infiltration. In summary, murine epidermis responds to an acute UV injury in vivo with an initial abrogation of antigen-presenting activity followed by epidermal infiltration with neutrophils, differentiated macrophages, and monocytic antigen-presenting cells that are distinct from Langerhans cells with regard to expression of Langerhans cell markers and ultrastructure.     

Fine-needle aspiration of histiocytic necrotizing lymphadenitis (Kikuchi's disease)

Fine-needle aspiration (FNA) of the lymph node was done in five patients with histiocytic necrotizing lymphadenitis (Kikuchi's disease). In four patients, the aspirates were found to have many small and large atypical lymphocytes, some reactive, phagocytic histiocytes, and intense extracellular debris. Neutrophils, plasma cells, or multinucleated giant cells were not seen. These cytologic findings were considered diagnostic for Kikuchi's disease. In one patient, the aspirate did not show significant histiocytosis or tissue necrosis and was considered nondiagnostic. In patients with both typical clinical features and characteristic cytologic findings in the lymph node aspirates, FNA of the lymph node alone will suffice for diagnosis. In those patients with typical clinical features but nondiagnostic findings in the FNA aspirates, the diagnosis of Kikuchi's disease may have to be established either on repeated nodal FNA or on lymph node biopsy.     

Induction of antigen presentation by macrophages after phagocytosis of tumour apoptotic cells

Due to their resistance to classical chemotherapies, most human colorectal cancers have a high incidence and a poor prognosis. Immunotherapy using interleukin 2 (IL2) has provided disappointing results in the treatment of these cancers. Recently, however, we have demonstrated that a treatment combining a cell-differentiating agent, sodium butyrate (NaBut) with IL2 resulted in a remission of established peritoneal colorectal carcinomatosis in rats. Separately, neither NaBut nor IL2 treatment cured these tumour-bearing rats. NaBut is known to induce cell differentiation and subsequent apoptosis in epithelial cells, while IL2 stimulates the immune cells capable of participating in tumour rejection. We postulated that the significant therapeutic effect of NaBut/IL2 treatment could be attributed to a NaBut-induced increase in the immunogenicity of the cancer cells. We report here that NaBut induced an apoptotic process in rat colon tumour cells in vivo and in vitro. We observed, in an efficient cure, colocalization of apoptotic bodies and monocytes/macrophages at the periphery of the tumour. We propose that these apoptotic bodies are phagocytosed in vivo by the macrophages. We also showed in vitro that a subpopulation of macrophages involved in the phagocytic clearance of apoptotic cells expresses cell surface molecules associated with antigen presentation and stimulates the proliferation of naive splenocytes. Our data suggest that therapies that recruit massive induction of the apoptotic process in tumour cells could favour tumour antigen presentation via their specific phagocytosis by antigen-presenting cells (APCs). We propose that the development of specific therapies that stimulate both tumour cell apoptosis and the immune system could offer new opportunities in anti-cancer treatments of poorly immunogenic cancer cells.     

Modulation of macrophage function for defence of the lung against Pseudomonas aeruginosa

Pseudomonas aeruginosa is a common respiratory tract pathogen in certain groups of compromised hosts, most notably those with cystic fibrosis. The pathogenicity of P. aeruginosa may depend in part upon its capacity to resist normal phagocytic cell clearance. We have recently shown that phagocytosis of P. aeruginosa by macrophages is a unique two-step process; binding is glucose-independent but ingestion occurs only in the presence of D-glucose or D-mannose. P. aeruginosa is the only particle we have found which is ingested by macrophages in a glucose-dependent manner. Since glucose is present in only negligible quantities in the endobronchial space, P. aeruginosa may be pathogenic by virtue of its capacity to exploit the opportunity presented in the lower airway to resist normal nonspecific phagocytic defences. The purpose of the studies reported here is to better understand the glucose-dependent phagocytosis of P. aeruginosa and to design novel therapies to facilitate phagocytic cell clearance of it from the lower respiratory tract. We have shown that phagocytosis of unopsonized P. aeruginosa depends upon facilitated transport of glucose into macrophages via the GLUT1 isoform. After transport into the macrophage, the glucose must be metabolized to trigger phagocytosis of P. aeruginosa; pretreatment with 2-deoxyglucose or 5-thioglucose abrogates glucose-dependent ingestion. We have recently demonstrated that pulmonary alveolar macrophages (as opposed to all other macrophage phenotypes studied) lack the capacity to transport glucose and to phagocytose unopsonized P. aeruginosa; however, after the cells have been cultured in vitro for 48 hours, they are able to perform both functions. Whereas most macrophages (such as peritoneal cells) primarily depend upon glycolysis for metabolic energy, pulmonary alveolar macrophages reside in a high oxygen tension environment and appear to utilize oxidative phosphorylation. Treatment of freshly explanted pulmonary alveolar macrophages with sodium azide (to poison oxidative respiration) dramatically enhances both glucose transport and glucose-dependent phagocytosis of P. aeruginosa. We are currently investigating the compromised phagocytic function of pulmonary alveolar macrophages and the mechanism by which azide enhances glucose transport and phagocytosis of P. aeruginosa. Although physiological measurements have indicated that glucose is removed from the endobronchial space by an active transport process of the lung epithelium, the types of glucose transporters that are expressed in the lung are as yet unknown. Using RT-PCR, we have amplified a product from human and murine lung RNA which has a high degree of homology with members of the sodium-dependent glucose transporter (SGLT) family. The ultimate goal of these studies is to design novel agents for enhancing the phagocytic function of pulmonary alveolar macrophages. Delivery of simple glucose by aerosol would not be effective because (i) it would be exported by sodium-dependent active transport and (ii) pulmonary alveolar macrophages lack the capacity to transport glucose. Various approaches for targeting glucose to alveolar macrophages by receptor-mediated endocytosis are under investigation.     

Immune adherence reactivity of rat alveolar macrophages following inhalation of crocidolite asbestos

The immune adherence phenomenon was used to demonstrate the in vivo deposition of complement on membranes of alveolar macrophages from rats chronically exposed to crocidolite asbestos dust. Pre-treatment of macrophage cualtures with anti-C3 antiserum greatly diminished the level of immune adherence reactivity. Alveolar macrophages exposed to crocidolite asbestos in vitro did not exhibit significant levels of immune adherence reactivity. These results may reflect an in-vivo antigen-antibody-complement interaction on the surface of a alveolar macrophages from animals which have inhaled asbestos dust.