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Although platelet factor 4 (PF-4) and the beta-thromboglobulin (beta-TG) proteins represent the first chemokines to be discovered, their functional roles in host defense became clear only recently. Residing in platelets as storage proteins and becoming released into the blood at very high concentrations, these mediators appear to fulfill different and complementary tasks as first-line mediators in the recruitment and activation of leukocytes, as well in the regulation of tissue repair. Whereas both proteins are structurally closely related members of the CXC chemokine subfamily, they are subject to quite dissimilar regulatory mechanisms controlling their generation and their spectrum of biological activities. Thus, proteolytic processing of inactive precursors plays a decisive role in whether the beta-TG proteins will act as stimulatory or inhibitory agents in neutrophil activation via the G protein-coupled receptors CXCR-1 and 2. PF-4, existing as a single molecular form, is largely resistant to proteolytic modification, but its interaction with an unusual receptor(s) on leukocytes (a proteoglycan) appears to depend on its oligomeric state. There is growing evidence that both chemokines may interfere with each other at various regulatory levels to promote coordinated cell activation. Moreover, recent findings suggest novel and unexpected activities for these chemokines, which may extend our view on early host defense.

Tumor regression induced in cancer patients by local instillation of bacillus Calmette-Guérin (BCG) into the bladder is considered to be mediated by cellular immune and inflammatory reactions. In an attempt to elucidate which of these effects are relevant to tumoricidal activity, an in vitro system was employed in which the immunostimulatory effects of BCG could be studied. This report describes the induction of BCG-activated killer (BAK) cells, which effectively lyse bladder tumor cells. Human peripheral blood mononuclear cells (PBMC) were stimulated with viable and sonicated BCG (v-BCG and s-BCG, respectively) to generate BAK cells. Cytotoxicity of BAK cells was comparable with the cytotoxicity exerted by lymphokine-activated killer (LAK) cells generated by interferon (IFN)-gamma but did not reach the level of interleukin-2 (IL-2)-generated LAK cells. Induction of BAK cells was possible only with v-BCG and not with s-BCG. By depletion and enrichment of defined cell populations, the cytotoxic potential of BAK cells could be attributed to a population of CD8(+) and CD56(+) double-positive lymphocytes. Macrophages and CD4(+) cells were required for the induction of killing activity but had no such activity by themselves. Furthermore, the presence of IFN-gamma and IL-2 in the supernatants harvested during the generation of BAK cells was demonstrated. Monoclonal antibodies neutralizing these cytokines abolished BCG-mediated cytotoxicity. From these results, it is concluded that the known beneficial effect of local instillation of BCG on maintenance of the relapse-free state in superficial bladder cancer may be due to local generation of BAK cells.

The activation of immunocompetent cells by lipopolysaccharide (LPS) during severe Gram-negative infections is responsible for the pathophysiological reactions, possibly resulting in the clinical picture of sepsis. Monocytes recognize LPS mainly through the LPS receptor CD14, however, other cellular binding structures have been assumed to exist. In previous studies, we have described an 80-kDa LPS-binding membrane protein (LMP80), which is present on human monocytes as well as endothelial cells. Here we demonstrate that LMP80 is widely distributed and that it formes complexes together with LPS and sCD14. Furthermore, we report on the biochemical purification of LMP80 and its identification as decay-accelerating factor, CD55, by amino acid sequencing and cloning techniques. Our results imply a new feature of CD55 as a molecule which interacts with LPS/sCD14 complexes. However, the involvement of CD55 in LPS-induced signaling remains to be elucidated.

In this study we describe a general method for the detection and characterization of endotoxin-(lipo-polysaccharide, LPS)-binding membrane proteins. In the past, experimental procedures to detect LPS-binding sites on cells were generally performed with chemically modified LPS derivates. Since anymodification of a ligand may lead to a modification of its binding characteristics, the results of thosestudies are controversial.In our assay, cell membrane preparations are treated with free lipid A, the endotoxic center of LPS,in the presence of normal human serum. After binding of lipid A, membrane proteins are solubilized bymild detergent treatment without disruption of the lipid A-protein complexes. Addition of anti-(lipid A)mAbs and subsequent adding of protein A agarose lead to the precipitation of complexes of lipid A andits binding proteins. By SDS/PAGE and western blot, these precipitates can be screened for the presenceof LPS/lipid A-binding proteins. We describe the use of this method for the immuno-coprecipitation oflipid A (or LPS) with an 80-kDa LPS-binding membrane protein (LMP80), which we have previouslyidentified on several human cells. In addition, CD14, the well-known functional LPS receptor on mono-cytes and macrophages, can be detected.By means of this immuno-coprecipitation approach we could demonstrate binding of either purifiedLPS preparations or synthetic lipid A to these LPS/lipid A-binding membrane proteins at physiologicalpH under conditions in which the proteins are in their natural membranous environment.Keywords: lipopolysaccharide; lipopolysaccharide-binding protein; CD14; anti-lipopolysaccharide anti-body; 80-kDa lipopolysaccharide-binding membrane protein (LMP80).Lipopolysaccharides (LPS, endotoxin) constitute the major biological activity and, therefore, a putative LPS-binding mole-component of the outer membrane of gram-negative bacteria. cule should also bind lipid A. In the immuno-coprecipitationLPS are responsible for many pathophysiological effects ob- assay we incubated cell membrane preparations, which containserved during gram-negative septic episodes [1, 2]. The LPS of the proteins in their native conformation, with lipid A. Afterall families of gram-negative bacteria contains the highly con- binding of lipid A to the LPS-binding molecules, the cell mem-served lipid part termed lipid A, which is able to mimic the branes were washed several times and solubilized by mild de-biological effects of LPS [1, 3]. Thus, lipid A represents the tergent lysis without disruption of the lipid A-protein complexes.endotoxic center of LPS. These complexes were precipitated by addition of anti-(lipid A)Many experimental procedures for the detection of the cellu- mAbs together with protein A agarose beads. After some wash-lar targets of LPS in host cell membranes have been carried out ing steps the precipitates were eluted by adding SDS sampleunder non physiological circumstances: Either the LPS prepara- buffer (62.5 mM Tris, 80 mM dithiothreitol,10% glycerol, 2%tion was chemically modified by labeling with or substitution SDS, 0.002% Bromophenol blue, pH 6.8) and separated by SDS/for a cross-linker, or the proteins under investigation were not PAGE. Specific proteins were detected by an immunoblot tech-in their physiological conformation after SDS/PAGE and west- nique on nitrocellulose membrane. The immuno-coprecipitationern blot techniques. Modification of a receptor molecule or its method not only leads to the detection of LPS-binding proteins,ligands for binding studies is critical, as the modification may it also permits the enrichment of those molecules from cellhave a strong influence on the binding behaviour. Therefore, the membrane preparations.physiological relevance of many molecules found by the use of Here, we demonstrate the successful use of this method bythose techniques remains controversial. the identification of lipid A binding to an 80-kDa LPS-bindingTo obtain a more physiological detection system for LPS- membrane protein (LMP80), which was previously described bybinding molecules we developed an immuno-coprecipitation our group [4] as well as to the LPS receptor CD14. Furthermore,assay with free lipid A and anti-(lipid A) antibodies. Lipid A we examined the binding of LPS as well as synthetic oligoacylwas used, because it is the minimal structure of LPS with full lipid A partial structures to LMP80 [527].

Colony-stimulating factors (CSF) are used clinically in the treatment of chemotherapy-induced myelosuppression and in support of bone marrow transplantation. As CSF are known to have pleiotropic functions, their effects on cellular cytotoxicity were analysed in vitro against bladder carcinoma cell lines. By means of an L-[3H]methionine-release assay, the cytotoxicity of peripheral blood mononuclear cells against the natural-killer(NK)-cell-resistant bladder carcinoma cell lines BT-A and SBC-7 was measured using different effector/target-cell ratios. Costimulatory effects of granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and stem cell factor (SCF) on the generation of lymphokine-activated killer (LAK), bacillus Calmette-Guérin-activated killer (BAK) and natural killer (NK) cell cytotoxicity were investigated in this assay. Furthermore, the effect of CSF on proliferation of urothelial tumor cells in vitro was determined by a [3H]thymidine DNA-labelling technique. GM-CSF, but not G-CSF, IL-3 or SCF, was able to increase NK, BAK and LAK cytotoxicity in a dose-dependent manner. No acceleration of carcinoma cell proliferation was evident under the conditions of our assay. These data indicate the costimulatory effect of GM-CSF on cellular cytotoxicity, which might be used for immunotherapeutic purposes.

The interaction of CD28 with its ligands is important for T-cell activation. Recent studies demonstrated the existence of at least two ligands on accessory cells, CD80 (B7-1) and CD86 (B7-2). In this study we demonstrate that, although CD80 and CD86 are both expressed on monocytes, they seem to have different functions. Freshly isolated monocytes express CD86 but are CD80-negative. CD80 expression is weakly induced after 6-8 hr of in vitro culture and is enhanced by stimulation. CD86 expression is enhanced faster than CD80 expression and reaches the peak level after 4-6 hr in stimulated cells. Reverse transcription-polymerase chain reaction studies demonstrate that freshly isolated monocytes contain no CD80-mRNA. The mRNA of CD80 is induced after 4-6 hr of culture, which matches with the expression of the protein. Inhibition studies using different antibodies against both molecules and the fusion protein CTLA4Ig show that only anti-CD80 and CTLA4Ig could partially inhibit antigen-specific (tuberculin) and polyclonal (anti-CD3) lymphoproliferation and interferon-gamma (IFN-gamma) secretion of T cells cocultured with autologous monocytes. IFN-gamma secretion was more sensitive to blocking costimulation than proliferation. The antibody BB-1 did not inhibit proliferation and cytokine secretion, nor did the anti-CD86 clone IT2.2. CTLA4Ig, which binds both CD80 and CD86, has the same inhibitory capacity as the anti-CD80 antibody tested. From those findings we conclude that human monocytes use CD80 as a costimulatory ligand for CD28 and utilize other costimulatory mechanisms besides those mediated via molecules of the B7 family.

To detect mRNA expression of nitric oxide synthase (NOS) isoforms in human monocytes/macrophages reverse transcription polymerase chain reaction (RT-PCR) was used. mRNA was isolated from stimulated or unstimulated monocytes/macrophages and RT-PCR was performed using oligonucleotide primers derived from mRNA sequences of either human endothelial constitutive (c) or human hepatocyte inducible (i) NOS. RT-PCR of mRNA isolated from resting monocytes and macrophages resulted in the amplification of a cNOS specific mRNA fragment. When the cells were stimulated with lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) prior to mRNA extraction, RT-PCR yielded an iNOS-specific amplification product. Whereas the activation of both cell types was accompanied by expression of iNOS mRNA, the cNOS signal seemed to be diminished upon immunostimulation. Not only in purified human monocytes but also in the human monocytoid cell lines MonoMac 6, THP-1, and U937 cNOS mRNA was detected. The data clearly demonstrate the presence of iNOS and cNOS mRNA in human monocytes/macrophages and provide the necessary tools to investigate the regulation of NO synthesis in these cell populations.

Fc gamma receptor I-positive (CD64+) and Fc gamma receptor I-negative (CD64-) monocytes were prepared from highly purified (elutriation-derived) human monocytes by cytofluorograph cell sorting, and a phenotypical and functional dissociation of the isolated CD64+ and CD64- monocyte subsets is demonstrated. Surface analyses revealed that the surface antigen pattern of CD64+ monocytes corresponds to the phenotype of typical unseparated monocytes. In contrast, CD64- monocytes are characterized by high expression of major histocompatibility complex (MHC) class I antigens (HLA-A, -B, -C) and MHC class II antigens (HLA-DR, -DP, -DQ), and low expression of the monocyte-specific marker CD14 which is found on nearly all CD64+ monocytes. However, 75% of the CD64- cells were found to be esterase-positive, and 85% were positive for the the CD64- cells were found to be esterase-positive, and 85% were positive for the monocyte/macrophage-specific intracellular antigen CD68. Furthermore, CD64- monocytes show significantly higher expression of CD45RA and Fc gamma receptor III (CD16) than CD64+ monocytes, but lack the natural killer cell markers CD56 and CD57. Functional studies showed that cells of the minor CD64- monocyte subset have a higher accessory cell capacity in antigen-driven T cell activation than CD64+ monocytes. CD64- monocytes pretreated with PPD (purified protein derivative of tuberculin) induced up to tentimes more interferon-gamma and also higher proliferation in responding autologous T cells than PPD-pretreated CD64+ monocytes. Similar results were obtained for T cells in mixed leukocyte reaction. Interferon-gamma release and proliferation of allogeneic lymphocytes were consistently higher in the presence of irradiated CD64- monocytes than of irradiated CD64+ monocytes. Furthermore, when CD64- and CD64+ monocytes were stimulated with Newcastle disease virus, we measured an up to 67-fold higher interferon-alpha release from CD64- than from CD64+ monocytes, indicating a higher anti-viral capacity of this subset. CD64- monocytes showed lower activity in the phagocytosis of unopsonized particles and also lower zymosan- or latex-induced chemiluminescence than CD64+ monocytes. These findings indicate that CD64- monocytes, although comprising only less than 10% of all peripheral blood monocytes, represent a monocyte subpopulation efficiently interacting in vitro with T cells and, additionally, are the major source of interferon-alpha.

Intragraft production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1-beta) was determined in rejecting human liver grafts during acute rejection and in chronic graft dysfunction. The localization of cytokine-producing cells was then correlated with the distribution of monocytes and macrophages as their main producers, as well as with effector functions such as endothelial cell activation. In selected patients collateral TNF-alpha plasma levels were measured. In normal liver and biopsies taken during an uncomplicated course, few TNF-alpha and even fewer IL-1-beta positive macrophages were found. During acute rejection episodes of all degrees of severity liver grafts were infiltrated by large numbers of TNF-alpha-positive monocytes, and concomitant TNF-alpha plasma levels were elevated compared with uncomplicated controls. In marked contrast IL-1-beta production by macrophages and vascular and sinus endothelial cells was restricted to the most severe, irreversible rejection episodes. The localization of cytokine-positive cells coincided with areas of maximum induction of ICAM-1 and von Willebrand Factor. In chronic graft dysfunction increased numbers of mature macrophages were found. A large proportion of these were positive for TNF-alpha as well as IL-1-beta. Distinct from acute rejection episodes, however, parallel TNF-alpha plasma levels were not elevated, suggesting cytokine storage rather than secretion. The present results indicate an important local role of TNF-alpha and IL-1-beta in the early phase of the rejection process. They presumably activate endothelial cells to upregulate the expression of adhesion molecules, thereby facilitating mononuclear cell adhesion and extravasation. Therefore, specific inactivation of cytokines or of their actions may prove to be a powerful tool in the prevention and treatment of allograft rejection in the future.

The effects of synthetic alkyl ((alkyl 6-deoxy-a-D-gluco-heptopyranosyluronate) 6-deoxy-a-D-gluco-heptopyranoside) uronates, a novel type of mirror pseudo cord factor, on the in vitro modulation of interleukin-6 production and T-cell proliferation in human peripheral blood mononuclear cells, were investigated. Synthetic mirror pseudo cord factors with alkyl chains ranging from C16 to C18 have very weak interleukin-6-inducing capacities and lack mitogenic activities for T-cell proliferation. However, they could inhibit IL-6 release induced by sonicated Bacillus Calmette-Guérin (S-BCG), bacterial endotoxin, and phytohaemagglutinin in a dose-dependent manner. Inhibition was observed not only with mononuclear cells but also with purified monocytes. Furthermore, these synthetic compounds could suppress T-lymphocyte proliferation stimulated by sonicated Mycobacterium tuberculosis H37Rv (S-H37Rv) antigens, S-BCG antigens, as well as by recombinant 65 kDa mycobacterial heat-shock protein. In contrast, these compounds failed to inhibit the phytohaemagglutinin-induced T-cell proliferation. We conclude that the inhibition of cytokine release and T-cell proliferation by synthetic mirror pseudo cord factors was due to direct blocking of the function and/or activity of monocytes or antigen-presenting cells.

Bone-marrow transplantation (BMT) from an unrelated, HL-A-phenotype-identical, MLC-negative donor was performed in a 31 year old woman with severe longlasting aplastic anemia. In vitro assays failed to demonstrate humoral or cellular sensitization of the recipient against donor-type antigens. Following conditioning with cyclophosphamide, prompt but only transient engraftment of the transplant occurred accompanied by signs of mild graft-versus-host-disease (GVHD) of the liver. The results of a second bone marrow transplantation from the same donor cannot be evaluated due to early death of the recipient. It is concluded that bone marrow from unrelated, HL-A and MLC-identical donors may engraft without severe GVHD. Rejection of the graft in our patient may have been related to greater antigenic differences that can be expected to exist between HL-A and MLC-identical unrelated individuals than between HL-A and MLC-identical siblings. However, insufficient preparative immunosuppression with cyclophosphamide due to severe hepatic hemosiderosis appears equally likely as the cause of graft rejection. The possibly increased risk of graft rejection or severe GVHD should not preclude the use of unrelated HL-A and MLC-identical marrow donors, when histocompatible sibling donors are not available; but more potent immunosuppressive regimens than the cyclophosphamide protocol may be necessary to ensure permanent engraftment.

Despite the lack of knowledge about its mode of action, immunotherapy of leukaemia continues to be a major challenge for physicians and experimental oncologists. Basic research should be aimed at defining leukaemia-associated antigens in humans and developing test systems for the detection of immune responses against leukaemia. Clinical trials are only valid when designed in a strictly randomized controlled fashion. Whether we can expect further advances in the field of immunotherapy will ultimately depend on sound scepticism as well as on new and unconventional ideas of the investigator.

To analyze the interaction between macrophages and splenic lymphocytes with reference to time and concentration, the Mishell-Dutton system was divided into two experimental steps. Step 1 consisted of the cocultivation of spleen cells with various doses of macrophages for different periods of time, while in step 2 macrophages were removed, spleen cells transferred to fresh petri dishes and cultivated until plaque assay. Cocultivation of spleen cells with high doses of macrophages for 4--8 h markedly enhanced the DNA synthesis and plaque-forming cell (PFC) response of sheep red blood cell-stimulated and unstimulated cultures. A cocultivation longer than 24 h resulted in an inhibition of both DNA synthesis and PFC response of spleen cells. These studies suggest a nonspecific function of macrophages on proliferation and differentiation processes in antibody formation.

The correlation between MLC reactivity (LD) and serological leukocyte typing (SD) was studied in a beagle colony. Disparity for a serologically defined non-DL-A lymphocyte antigen did not correlate with MLC reactivity. Lymphocytes of colony members with common ancestors and SD identical DL-A haplotypes did not stimulate each other in the MLC. This implies that LD typing in the beagle coolony can be generally predicted by DL-A SD typing. Consequently, lymphocytes of sibs homozygous for a given DL-A SD haplotype could be shown, with few exceptions, to be also homozygous for MLC determinants. Cells of these homozygous sibs can be used in MLC typing as reference cells for DL-A LD specificities. Two exceptions to the expected linkage between DL-A SD typing and MLC reactivity were found. These findings could not be explained by recombination with the DL-A region assuming a single major LD locus coding for MLC. Thus, suggestive evidence for more than one single LD locus has been obtained.

In the presence of serum, 2-mercaptoethanol-treated bovine serum albumin enhances T cell colony formation as does 2-mercaptoethanol. The factor only partially substitutes for FCS, but neither for the mitogen phytohemagglutinin nor for conditioned medium derived from cultures of adherent cells.

Durch die Anwendung des Multitest-Hautstempels vor einer operativen Intervention wurde bei Patienten mit soliden Carcinomen die Immunreaktion vom verzogerten Typ untersucht. Es zeigt sich, das die Rate an positiven Testen in einem inkurablen Tumorstadium deutlich reduziert ist im Vergleich zu Patienten mit potentiell kurablen Tumorstadien sowie zu Kontroll-Patienten mit nicht-malignen Erkrankungen. Bei Patienten in einem fortgeschrittenen Tumorstadium finden sich wesentlich haufiger anerge und hyperge Reaktionslagen als bei potentiell kurablen Tumorpatienten sowie Nicht-Malignom-Patienten. Lokale oder systemische Nebenwirkungen konnten bei keinem der 86 untersuchten Patienten beobachtet werden.