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Dendritic cells (DC) modulate immune responses depending on the nature of the antigens. Receptors capable of discriminating these antigens on the basis of the pathogen-associated molecular patterns (PAMP) belong to the Toll-like receptor (TLR) family. The macrophage-activating lipopeptide 2 kDa (MALP-2), a synthetic lipopeptide derived from Mycoplasma fermentans, signals through TLR-2 and TLR-6. The aim of this study was to examine whether MALP-2 can modulate the functional properties of human monocyte-derived DC. The effects of this treatment were compared to those of the TLR-4 agonist lipopolysaccharide (LPS). To ensure clinical applicability, DC were generated under serum-free conditions. MALP-2 and LPS stimulation induced the expression of CD83 and increased the expressions of CD80, CD86, HLA-ABC and CD40. Furthermore, both substances decreased the endocytotic capacity of DC and induced the release of bioactive TNF-alpha and IL-10, whereas LPS additionally increased IL-12 release. Pretreatment with both substances boosted the allostimulatory capacity of DC. In a coculture with autologous lymphocytes, either MALP-2 or LPS pretreated DC induced a marked proliferation of lymphocytes, but only DC prestimulated with MALP-2 activated lymphocytes to produce the cytokines IL-4, IL-5 and IFN-gamma. No polarisation of lymphocytes into T-helper (Th)1 or Th2 was detected. These data indicate that MALP-2 is a potential candidate to modulate DC for clinical applications.

The capacity of intrinsic, glomerular mesangial cells (MC) to cause an autoreactive response of syngeneic lymphocytes in vitro are presented. Initial experiments demonstrated the MHC class II dependent capacity of MC to present exogenous antigen to sensitized lymph node lymphocytes (LN) and to activate naive, allogeneic LN in the absence of a nominal antigen. However, the most striking finding of the present investigation was that mouse MC (C57BL/6 or DBA/2) augmented a significant activation of naive, syngeneic lymphocytes. The extent of the proliferative lymphocyte response was comparable to that observed after stimulation with allogeneic MC. Moreover, during syngeneic coculture substantial amounts of interferon bioactivity were generated. Equipotent concentrations of rm IFN-gamma were sufficient to induce class II MHC expression of mouse MC. In control experiments the macrophage cell line, IC-21 (C57BL/6), or freshly prepared DBA/2 mouse peritoneal macrophages did not elicit a syngeneic LN response. Using MC, which had not been pretreated, the MC-specific LN stimulation occurred after prolonged periods of coculture. The stimulation index (S.I.) was 9.77 after 144 hours compared with LN controls (S.I. = 1). However, a 48 hour pretreatment of MC with either rm IFN-gamma alone or in combination with rh TNF-alpha and/or the continuous presence of rm IL-1 alpha during coculture periods from 72 to 144 hours substantially enhanced the proliferative LN response. Analysis of non-adherent LN by flow cytometry (FACS) after 96 or 120 hours coculture with MC revealed an increased ratio of Thy1.2+ to B220+ cells with a predominant rise of L3T4+ T-helper cells compared to Lyt2+ cytotoxic T-cells. Furthermore, immune fluorescence microscopy showed that a fraction of Thy1.2+ lymphoblasts adhered to MC. FACS analysis of these adherent LN after detachment demonstrated that in comparison to cocultures with untreated MC, cocultures of LN with IFN-gamma/TNF-alpha pre-treated MC resulted in a 24.4% increase of Thy1.2+ cells, with 89% of these being L3T4+ T-helper lymphocytes. In conclusion, autoreactivity of preferentially T-helper cells to cocultured glomerular MC was shown, which may represent a useful model of T-lymphocyte dependent glomerulonephritis.

Analysis of the functional activity of phagocytes is of great importance in the differential diagnosis of patients with recurrent bacterial infections. Here we describe a method to determine the production of reactive oxygen intermediates (ROI) by microcytofluorometry using dihydrorhodamine 123, a derivative of rhodamine 123. Using this method the ROI production of erythrocyte-depleted whole blood samples can be measured without further time-consuming purification steps. Possible harmful manipulation of the isolated cells can also be avoided and highly reproducible and significant results are obtained in the minimum of time. This assay provides a very sensitive alternative to the clinically used NBT test in the diagnosis of patients with chronic granulomatous disease (CGD). Moreover, the analysis of oxygen-dependent effector functions of murine effector cells and cell lines may be important in investigating resistance to certain microbes (e.g., Candida albicans, Staphylococcus aureus or different protozoa such as Toxoplasma gondii or Leishmania species).

C57BL/6 macrophage populations from spleen and liver, the main organs for the manifestation of visceral leishmaniasis, were investigated for their ability to perform spontaneous phagocytosis-associated killing of 3H-thymidine (3H-TdR)-prelabelled L. donovani amastigotes and promastigotes. The results showed that organ macrophages from spleen and liver killed L. donovani amastigotes and promastigotes spontaneously with high efficiency. This consistent finding was first detectable at 2-3 h, and the reaction was completed at 12 h. This type of killing was strongly enhanced when spleen and liver macrophages were activated. This phagocytosis-associated killing mechanism may contribute, to a large extent, in maintaining the infection under control in vivo, by drastically reducing the amount of parasites that is required to establish intracellular parasitism. To be able to assay phagocytosis-associated destruction of both promastigotes and amastigotes, a reproducible system for the production in vitro of Leishmania donovani amastigotes by the macrophage cell-line J774 was developed. The DNA of the Leishmania amastigotes was labelled with 3H-TdR with high efficiency. The spontaneous label release of prelabelled L. donovani amastigotes was comparable to that of prelabelled promastigotes over an assay period of 24 h.