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Cutaneous leishmaniasis is initiated by the bite of an infected sandfly and inoculation of Leishmania major parasites into the mammalian skin. Macrophages are known to playa central role in the course of infection because they are the prime host cells and funetion as antigen-presenting eells (APC) for induetion of the eell-mediated immune response. However, in addition to maerophages in the dermis. the skin eontains epidermal Langerhans eells (LC) which ean present antigen (Ag) to T cells. Therefore, using a murine model of cutaneous leishmaniasis, we analyzed the ability of epidermal cells to induce a T eell response to L.major. The results demonstrated that freshly isolated LC, but not cuItured LC, are highly active in presenting L.major Ag in vitro to T cells from primed mice and to a L.major-specific T cell clone. Furthermore, freshly isolated LC had the ability to retain L.major Ag in immunogenic form for at least 2 days. Their efficiency was much greater than that of irradiated spleen cells, a standard population of APC. LC stimulated both T cell proliferation and production of the Iymphokines interleukin (IL)-2 and IL-4. The response was Ag specific and could be induced by lysate of L. major parasites and by live organisms. The data suggest that epidermal LC are important APC in eutaneous leishmaniasis. They may perform a critical funetion by eapturing L.major Ag in the skin and presenting it either to quiescent T eells circulating through the draining lymph node or locally to T effector cells infiltrating the cutaneous lesion.
Die Unterscheidung zwischen körpereigenen und körperfremden Strukturen ist eine grundlegende Herausforderung der spezifischen Immunantwort. Pathologische Veränderungen dieser Abgrenzung können zu schwerwiegenden Autoimmunerkrankungen wie beispielsweise Diabetes Mellitus, Rheumatischer Arthritis oder Multipler Sklerose führen. Um unerwünschte (Auto-) Immunreaktionen zu verhindern, existieren verschiedene Formen von peripheren Toleranzmechanismen, die durch viele Transkriptionsfaktoren wie z. B. ICER (inducible cAMP early repressor), NFAT (nuclear factor of activated T cells) und Foxp3 (forkhead box protein p3) kontrolliert werden. Foxp3+ regulatorische T-Zellen (Tregs) sind spezialisierte immun-suppressive Lymphozyten, welche die Aktivierung anderer Immunzellen unterdrücken können. Einer der möglichen Mechanismen ist der Transfer zyklischen Adenosin-Monophosphats (cAMP) von Tregs in konventionelle T- und B-Lymphozyten. Die erhöhte intrazelluläre Konzentration an cAMP führt in Effektorzellen zur Induktion und Kerntranslokation von ICER. Der transkriptionelle Repressor ICER supprimiert die Expression vieler NFAT-regulierter Gene und hemmt darüber hinaus die Induktion der NFATc1/αA-Isoform selbst. Diese Isoform wird speziell in pro-inflammatorischen Effektorzellen hochreguliert und ist maßgeblich an deren spezifischem transkriptionellen Programm beteiligt. Foxp3 ist ein zentraler Faktor für die Bildung und Funktion sowohl Thymus-generierter nTregs als auch peripher (TGFβ-) induzierter iTregs. Die Kontrolle des Foxp3-Gens wird in iTregs – überraschenderweise aber nicht in nTregs – durch NFAT-Faktoren reguliert. Allerdings hemmt Foxp3 durch eine negative Rückkopplung wiederum die Induktion und Aktivität von NFATc1/αA. Dies stellt somit ein weiteres Regulativ dar, wobei Foxp3 nicht nur die Plastizität, sondern auch die Funktion von immun-suppressiven T-Zellen steuert. Zusätzlich regulieren die verschiedenen NFAT-Faktoren auch die Antigen präsentierenden dendritischen Zellen (DCs). Während NFATc1 und NFATc2 die Differenzierung und Proliferation von DCs beeinflussen, reguliert NFATc3 deren Zytokinexpression und steuert indirekt auch die nachfolgende T-Zell-Immunantwort. Die Kontrolle der Genregulation in Immunzellen durch die Transkriptionsfaktoren ICER, NFAT und Foxp3 erfüllt somit spezifische Funktionen der Immunität, reguliert aber gleichzeitig wichtige Aspekte der peripheren Toleranz, um schädliche (Auto-) Immunreaktionen zu verhindern.
In the present study, an attempt was made to characterize the immunomodulating abilities of the cytostatic drugs cydophosphamide, ifosfamide, vinblastine, vincristine, procarbazine, dacarbazine, 6-mercaptopurine, methotrexate, 5-f/uor-uracil and adriamycine in a defined experimental model. Varying combinations of drug plus transplantation alloantigen, (C3H-lymphocytes) were injected into Balb/c mice at different time intervals in vivo. The resulting T-effector cell reactivity was determined in vitro with the microcytotoxicity assay on day + 5 for primary (r) and day + 7 for secondary (2°) sensitized mice. According to the type of drug (alkylating agent vs. vinca alkaloid vs. antimetabolite vs. cytostatic antibiotic), the dosage (20% LD50 vs. 60% LD50), the state of sensitization (r vs. 2° sensitized recipients), and the time of drug application in relation to the antigen treatment on day 0 (in varying steps from day -6 to day +4), so-called "pharmaconantigen- variation-effects" (PA VE) were established for each of the investigated drugs in form of reaction profiles. The results were as folIows: (1) For almost alt substances, characteristic reaction profiles involving immunostimulation and/or immunosuppression could be established. Similarities in the profiles of different substances made it possible to classify the drugs according to different reaction types. The reaction type however is not definitely correlated to the biochemical mechanism of drug action. (2) The PA VE are decisively inf/uenced by so me of the biological parameters, such as the time of drug application in relation to the antigen treatment and the state of sensitization but relatively !ittle by the dosage of the drug. (3) Considering the different processes occurring du ring primary and secondary immune responses, the PAVE may give hints for a distinct manipulation of the immunoregulation and thus information on the immunobiological mechanism of drug action.
Die Aktivierung der T Zelle bedarf der spezifischen Interaktion zwischen T Zelle und Antigen-präsentierender Zelle unter Ausbildung einer engen Anlagerung beider Zellmembranen („immunologische Synapse“) für Rezeptoren-Interaktionen und konsekutive Signaltransduktion. In dreidimensionaler Kollagenmatrix zeigte sich ein stereotypes, dynamisches Muster bei der Interaktion zwischen CD45RO-positiven humanen T Zellen und antigenpräsentierenden dendritischen Zellen. i) Die Kontaktaufnahme wurde stets über das Leading edge der T Zelle initiiert. ii) Beim dynamischen Kontakt wanderte die T Zelle polarisiert, mit vielen Richtungsänderungen und mit reduzierter Geschwindigkeit auf der DC-Oberfläche, nur unterbrochen von kurzen Stopp- und Abrundungsphasen. Der Uropod der T Zelle stand während der dynamischen Kontakts in kontinuierlicher Verbindung zur DC. iii) Die Loslösung der T Zelle von der DC war ein aktiver Prozess, der durch Interaktion der Vorderfront der T Zelle zu benachbarten Kollagenfasern eingeleitet wurde, gefolgt von der Lösung des Zellkörpers und des Uropods. Alternativ wurden Kontakte durch Uropod-mediierte Retention der T Zelle auf der DC-Oberfläche verlängert. Zur dynamischen molekularen Charakterisierung der Kontaktfläche wurde eine Methode zur Darstellung von Lipid-Rafts an lebenden Zellen in der 3D ECM mit BTRITC etabliert. Die Ergebnisse zeigen ein neues 3-Schritt-Konzept dynamischer und produktiver Interaktionen zwischen T Zelle und DC in vitro. Die assymetrische Kontaktzone impliziert distinkte Funktionen von Vorderfront und Uropod der T Zelle und definiert eine neuartige dynamische Kontaktform für die Signalübertragung zwischen beweglichen Zellen.
Macrophages are important effector cells of the innate and adaptive immune response and exert a wide variety of immunological functions which necessitates a high level of plasticity on the chromatin level. In response to pathogen-associated molecular patterns (PAMPs) or inflammatory signals macrophages undergo a process of cellular activation which is associated with morphologic, functional and biochemical changes. Toll-like receptors (TLR) are able to sense many different PAMPs. TLR4 is an important sensor for lipopolysaccharide (LPS) which elicits a major portion of the host’s inflammatory response through the activation of many different signaling pathways such as the NF-κB and the MAPK protein kinase pathways RASRAF- MEK-ERK, p38 and JNK. Polycomb group (PcG) proteins are well known chromatin modifiers which function in large complexes and are required to maintain chromatin structure in a transcriptionally repressed state. It has previously been shown that the PcG protein Bmi1 is phosphorylated by 3pK, a downstream effector kinase of the MAPK protein kinase pathways RAS-RAF-MEK-ERK, p38 and JNK. In this work I analyzed the role of Bmi1 as a downstream effector of MAPK signaling during macrophage activation. Unexpectedly a rapid up-regulation on the Bmi1 protein level was observed in bone marrow derived macrophages (BMDMs) after LPS treatment. The Bmi1 induction was associated with transient protein phosphorylation that occured downstream of MAPK signaling. LPS treatment of BMDMs in the absence of Bmi1 resulted in a pronounced increase of IL-10 secretion. This secretion of the anti-inflammatory cytokine IL-10 was associated with increased IL-10 mRNA levels. Furthermore, siRNA mediated knock down of Bmi1 in J774A.1 macrophages also resulted in elevated IL-10 mRNA levels in response to LPS. ChIP analysis revealed that Bmi1 binds to throughout the il-10 locus. Alternative activation of wild type BMDMs via concomitant TLR4 and FcγR activation which triggers high IL-10 expression is paralleled by an attenuated Bmi1 protein expression. These results identify Bmi1 as a repressor of IL-10 expression during activation of macrophages.
Dendritic cell-based vaccination is a well established technique for preventive and therapeutic instruction of the immune system where conservative vaccine formulations fail to cure or prevent diseases, respectively. Efficiency of this technique already was demonstrated in infectious diseases as well as for cancer in animal or human studies. Well controlled manipulation and antigen-loading of immature DC is most beneficial to this technique. But, time-consuming and cost-extensive procedures for preparation of DC precursors, expansion and stimulation of DC and inpatient administration are big disadvantages regarding vaccine development for pandemic infectious diseases that occur mainly in underdeveloped countries. Therefore vaccines are needed that are pathogen-tailored and able to induce equal immune responses as their DC-based vaccine models. For vaccination against Leishmania parasites such a DC-based vaccine is feasible and its efficacy to induce protective Th1-based immune responses was already demonstrated in several animal studies. But, one of our own studies indicated supportive activity of host cells exceeding the allocation of T cells to become activated by transferred DC. IL-12, an important cytokine for the induction of Th1-related immune responses, has to be produced by host cells. Therefore, the aim of this study was to investigate the mechanism of BMDC-based vaccination with regard to simplification of the vaccine formulation. Key questions that have been addressed are: Which cells process the information that is transferred by the injected DC and what are the key components of this information? Further more, it was looked at whether altered vaccine formulations are able to induce protective immunity and whether they share equal molecular mechanisms. The current paradigm of BMDC-based vaccination proposes direct interaction of transferred BMDC with host T cells. These BMDC have to be antigen-loaded for stimulation via antigen-peptide-MHC molecule-complexes and they have to be activated for proper co-stimulation of T cells. Here, this study demonstrates that neither activation for co-stimulation nor direct interaction with adequate MHC molecules is needed for the induction of protective immunity against infection with Leishmania-parasites. Disrupted antigen-loaded BMDC are able to induce protective immunity in BALB/c mice without pre-stimulation via CpG ODN. Beyond, if BMDC were used with a different MHC-background than recipient mice then the vaccine still would be efficient in terms of reduction of footpad swelling and parasite load in draining lymph nodes. Even more, DC-specific features are no key component that leads to protective immunity as vaccination with disrupted antigen-loaded MΦ shows equal properties than before mentioned vaccine formulations. Further more, it was found that host DC play a major role in transforming the incoming signal, received from transferred antigen-loaded DC, into Th1-related stimuli and Leishmania-antigen-specific T cell activation. Suspensions of disrupted antigen-loaded DC resemble a combination of laid off soluble molecules together with exosome-like vesicles that formed after disruption of membranes. Here it was shown that separation of the membranous and soluble fractions and subsequent transfer into BALB/c mice will lead to protection of these mice against infection with L. major promastigotes only if the membranous fraction is used as vaccine. More, this vaccine formulation takes advantage of easy storage at -80°C with no need of fresh production. This clearly demonstrates that the immunity-inducing principle of disrupted DC-based vaccination lies within the membrane enclosed fraction. On a molecular level, disrupted antigen-loaded DC induce Th1-related cytokines during vaccination and as response on pathogen encounter. In vivo assays revealed IL-12 production and antigen-specific T cell proliferation among splenocytes that were stimulated with disrupted antigen-loaded DC. Splenocytes of accordingly vaccinated mice produce tremendous amounts of IFNγ after stimulation with Leishmania parasites. In summary, disrupted antigen-loaded BMDC fulfil all characteristics of DC-based vaccination against Leishmania major. But, while purification of membranes of antigen-loaded DC and subsequent transfer to BALB/c mice leads to control of the disease in the animal model, only slight levels of Th1-related cytokines are seen in the in vivo assays. Whether this points towards a loss of vaccine activity on unseen levels or unknown sites where Th1-related immunity is induced by both, complete solution and purified membranes, still has to be determined.
As critical steps in the life cycle oJ measles virus (Mfl), the e.fficiency of uptake into and replication in susceptible host cells are governed by cellular determinants. Measles virus infections of cells of the human CNS are characterized by particular constraints imposed on v1:ral transcription and translation attenuating viral gene Junctions and thus contributing to the pathogenesis oJ MV persistence in these cells.
The expression of measles virus (MV) in six different permanent human glioma cell lines (D-54, U-251, U-138, U-105, U-373, and D-32) was analyzed. Although all celllines were permissive for productive replication of all MV strains tested, U-251, D-54, and D-32 cells spontaneously revealed restrictions of MV transcription similar to those observed for primary rat astroglial cells and brain tissue. In vitro differentiation of D-54 and U-251 cells by substances affecting tbe intracellular cyclic AMP Ievel caused a significant reduction of tbe expression of tbe viral proteins after 18, 72, and 144 b of infection. This pronounced restriction was not paralleled to a comparable Ievel by an inhibition of tbe syntbesis and biological activity in vitro of virus·specific mRNAs as sbown by quantitative Northem (RNA) blot analyses and in vitro translation. The block in viral protein syntbesis could not be attributed to tbe induction of type I interferon by any of tbe substances tested. Our findings indicate tbat down-regulation of MV gene expression in human brain cells can occur by a cell type-rlependent regulation of tbe viral mRNA transcription and a differentiation-dependent regulation of translation, botb of wbicb may be crucial for the establisbment of persistent MV infections in tbe centrat nervous system.
To probe into the functional properties of the major peripheral myelin cell surface glycoprotein P 0 , its ability to confer adhesion and neurite outgrowth-promoting properfies was studied in cell culture. Tothis aim, Po was expressed as integral membrane glycoprotein at the surface of CV -1 cells with the help of a recombinant vaccinia virus expression system. Furthermore, the immunoglobulin-like extracellular domain of P0 (P0 -ED) was expressed as soluble profein in a bacterial expression system and used as substrafe coated to plastic dishes or as competitor in cell adhesion and neurite outgrowth-promoting assays. The adhesion of P0 -expressing CV-1 cells to P0 -ED substrafe was specifically inhibitable by polyclonal Po antibodies (54% :t 6% ). In addition, the specific interaction between Po molecules could be reduced ( 49% ± 8%) by adding soluble P0 -ED to the culture medium, demonstrating that the homophilic inter~ction between recombinant Po molecules can be mediated, at least on one partner of interacting molecules, by the unglycosylated Ig-like domain. Substrate-coated p -ED also conferred adhesion and neurite outgrowth ability to dorsal root ganglion neurons with neurites of a mean length of about 150 ,_..m. This neurite outgrowth was specifically inhibitable by soluble P" (74% ± 14%) and P 0 antibodies (65% ± 9% ). These observations indicate that Po is capable of displaying two different types of functional roles in the myelination process of . peripheral nerves: The heterophilic interaction with neurons may be responsible for the recognition between axon and myelinating Schwann cell at the onset of myelination, whereas the homophilic interacton may indicate its roJe in the selfrecognition of the apposing loops of Schwann cell surface membranes during the myelination process and in the mature compact myelin sheath.
Human immunodeficiency virus (HIV-1) infection in the human brain Ieads to characteristic neuropathological changes, which may result indirectly from interactions of the envelope glycoprotein gp 120 with neurons and/or glial cells. We therefore investigated the binding of recombinant gp120 (rgp120) to human neural cells and its effect on int~acellular.s.ignallin~. Herewe pre~ent evidence that rgp120, besides binding to galactocerebroside or galactosyl-sulfatlde, spec1f1cally bmds to a protem receptor of a relative molecular mass of approximately 180,000 Da (180 kDa) pre~ent. on the CD4-negative glioma cells D-54, but not on Molt4 T lymphocytes. Binding of rgp120 to this receptor rap1dly 1nduced a tyrosine-specific protein kinase activity leading to tyrosine phosphorylation of 130- and 115-kDa p~oteins. The c~ncentration of intracellular calciumwas not affected by rgp120 in these cells. Our data suggest a novel Signal transduc1ng HIV-1 gp120 receptor on CD4-negative glial cells, which may contribute to the neuropathological changes observed in HIV-1-infected brains.
The effect of measles virus (MV) infection on mRNA expression and protein synthesis of cytokines in human malignant glioma celllines (0-54 and U-251) was investigated. Primary MV infections led in both celllines to the induction of interleukin-1 fJ (ll-1 (3), interleukin-6 (IL-6), interferon-(3 (IFN-fJ), and tumor necrosis factor-a (TNF-a). ln contrast, persistently infected astrocytoma lines continually produced IL-6 (two out of 12 lines high Ievels) and IFN-ß, whereas only 1 out of 121ines synthesized TNF-a and none IL-1ß. The pathways for induction of IL-1fJ and TNF-a expression were not suppressed by the persistent MV infection, since IL-1ß and TNF-a could be induced by external stimuli Jike diacylglycerol analog plus calcium ionophore. lnterestingly, persistently infected astrocytoma cells synthesized considerably higher Ievels of ll-1ß and TNF-a than uninfected cells afteradditional external induction. These results suggest that in the centrat nervous system (CNS) of SSPE patients a percentage of persistently infected astrocytes may continually synthesize IL-6 and IFN-ß, and in the presence of additional external stimuli, as possibly provided by activated lymphocytes, might ovarexpress the inflammatory cytokines IL-1 ß and TNF-a. This may be of pathogenetic significance in CNS diseases associated with persistent MV infections.
To investigate the influence of inflammatory cytokines on the potential of peripheral nerves to regenerate, we analyzed the effect of interferon-y (lFN-y) and tumor necrosis factor-a (TNF-a) on the ability of immortalized Schwann cells to mediate outgrowth of neurites from primary DRG neurons. We found that IFN-y and TNF-a synergistically inhibited the neurite outgrowth-promoting properties of the Schwann cells by spedfically dowllregulating myelin-associated glycoprotein (MAG) at the levels of mRNA and cell surface protein by approximately 60%. Antibodies to MAG inhibited the outgrowth of neurites on Schwann cells to the same extent as treatment with the two cytokines. Since MAG appears to be involved in both neurite outgrowth and myelination, our findings may provide evidence for a mechanism, by wh ich inflammatory cytokines interfere with Schwann cell-neuron interactions.
Murine spienie T lymphocytes display maximal cellular myc gene (c-myc) expression already 3 h after concanavalin A timulation and sub equent down-regulation before the onset of DNA syntbesis. Stimulation by leucoagglulinin in the prcsence or absence of interleukin 2 Ieads to only low initiaJ Ievels of c-myc-specific RNA which, however, increase later on. A similar pattero of c-myc expression is shown by the Lyt- 2+ T cell subpopulation stimuiated with eilher concanavalin A or leucoagglutinin in the prescncc of interleukin 2. Although eH]thyn1idine incorporation was identical, the leucoagglutinin-stimulated Lyt-2+ T cells werc void of any demon. trable c-mycspeci. fic RNA at 3 h post-stimulation. Thus, the kinetics of c-myc expression in mause T lymphocytes arenot at all uniform, but depend on the mitogen and the subpopulation. [n contrast, lcvel8 of c-rasH•-spccific R A wcre always low at early times, always increased towards tbe onset ofDNA synthesis and down-regulationwas not observed.
In dieser Arbeit wurde der Einfluss mesenchymaler Stammzellen (MSC) auf verschiedene T-Zellsubpopulationen in vitro untersucht. Dazu wurden Naive- und Nicht-Naive CD4+ T-Zellen aus humanen PBMCs von gesunden Probanden und Patienten mit Autoimmun-Arthritis bei rheumatischen Erkrankungen isoliert und im Beisein/in Abwesenheit von MSCs unter Th-17-polarisierenden Bedingungen kultiviert. Nach einer 6 Tage umfassenden Inkubationszeit erfolgte die flowzytometrische Bestimmung des Phänotyps, der Proliferation, der Apoptose, des Zytokinprofils und der Chemokinrezeptorexpression Naiver und Nicht-Naiver-CD4+ T-Zellen im Beisein/in Abwesenheit von MSCs. Die Phänotypen wurden als CD45RA+CD27+ Naive-, CD45RA-CD27+ Gedächtnis-, CD45RA- CD27- Effektor- und CD45RA+CD27- TEMRA-Zellen definiert und ihre jeweiligen prozentualen Anteile an allen CD4+ T-Zellen bestimmt. Nach Beurteilung der Proliferation und Apoptose, erfolgte die Analyse der IFNγ-, IL-17-, IL-9- und IL-13-Produktion für jeden der vier Phänotypen. Zusätzlich wurde der prozentuale Anteil an FoxP3+CD25+CD127- Tregs und deren IL-10-Produktion bestimmt. Abschließend erfolgte die Messung der CCR5-, CCR6- und CXCR3- Expression. Insgesamt konnte sowohl in der Naiven CD4+- als auch in der Nicht-Naiven CD4+ T- Zellfraktion eine Hemmung der Proliferation und Apoptose CD4+ T-Zellen durch MSCs gemessen werden. Zudem supprimierten MSCs die Produktion der Zytokine IFNγ, IL-17, IL-9 und IL-10 und steigerten teilweise die Produktion von antiinflammatorischem IL-13. In den vier untersuchten Phänotypen verhielt sich die Zytokinproduktion variabel und war bei CD45RA-CD27+ Gedächtnis- und CD45RA-CD27- Effektor-Zellen am größten. Der hemmende Einfluss der MSCs war auf diese beiden Phänotypen ebenfalls am stärksten ausgeprägt. CD45RA+CD27+ Naive- und CD45RA+CD27- TEMRA-Zellen produzierten in Kultur mit MSCs mitunter vermehrt proinflammatorische Zytokine. Analog zur Proliferation und Apoptose verminderten MSCs die Expression von CCR5, CCR6 und CXCR3 auf CD4+ T-Zellen. Die beschriebenen Effekte der MSCs konnten sowohl bei gesunden Probanden, als auch bei Patienten mit rheumatischen Erkrankungen nachgewiesen werden. Durch die Verwendung eines Transwell®-Systems konnte gezeigt werden, dass MSCs ihre Wirkung auf T-Lymphozyten nicht nur durch direkten Zell-Zell-Kontakt, sondern auch über lösliche Faktoren ausüben. Die Resultate dieser Arbeit verdeutlichen den immunsuppressiven Charakter der MSCs auf Naive und Nicht-Naive CD4+ T-Zellen unter Th17-polarisierenden Bedingungen in vitro. Jedoch zeigt die Analyse der Zytokinproduktion in den untersuchten T-Zell-Phänotypen, dass MSCs neben ihrer immunsuppressiven Eigenschaft die Zytokinantwort einzelner T-Zellphänotypen steigern können. MSCs scheinen daher am ehesten eine immunmodulatorische Rolle zu spielen, indem sie übersteigerte Immunreaktionen herabsetzen und bei Bedarf immunstimulierend wirken.
Expression of the neural cell adhesion molecule and polysialic acid during early mouse embryogenesis
(1994)
The expression of the neural cell adhesion molccule (N-CAM) and a 2-8 linked polysialic acid (PSA), whieh is believed to be predominantly expressed on N-CAM, was investigated during early embryonie development ofthe mouse (embryonic days 7.5 to 10.0). By immunoeytoehemistry, in tissue sections, N-CAM and PSA were not detectable at embryonie day 7.5 but were expressed in the prominent body regions such as somites, unsegmented mesoderm, developing heart, and neuroectoderm at embryonie day 8.0 N-CAM and PSA immunoreaetivities were always predominantly associated with tbe plasma membrane. No tissue could be detected which was positive for PSA but negative for N-CAM. In Western blot analysis of whole embryos, by contrast, only the lightly sialylated and PSA-negative 180 and 140 kD isoforms of N-CAM werc present at embryonie day 8.0 and strong expression of PSA-bearing, heavily sialylated N-CAM was not detectable before embryonie day 10.0. In Western blot analysis of N-CAM immunoaffinity purifled from whole embryos and digested with neuraminidase as weil as in Northern blot analysis, the 120 kD isoform of N-CAM or its eorresponding mRN A were not expressed in detectable amounts during the time period investigated.
In dieser Arbeit wurden im Versuchstier Ratte die Transmission von T.gondii über die Muttermilch und deren immunologische Konsequenzen analysiert. Es konnte gezeigt werden, dass sich Rattenmilch als labordiagnostisches Medium eignet, wobei der direkte visuelle Parasitennachweis mit dem Lichtmikroskop, trotz verschiedender Aufbereitungsmethoden erfolglos blieb. Auch die PCR eignete sich für unsere Versuche nicht als Nachweismethode. Als geeignte und sensitive Methode für den Parasitennachweis in Rattenmilch stellte sich die Anzucht auf humanen Vorhautfibroblasten (HFF-Zellkultur)dar, wobei bereits zwei Tachyzoiten, die in vitro zu Milch nicht infizierter Tiere gegeben wurden, ausreichten, um eine HFF-Zelle zu infizieren. Immunisierungsexperimente wurden durchgeführt, um die Frage zu klären, ob die im Serum von Jungtieren nachgewiesenen Toxoplasma-spezifischen Immunglobulinen über die Muttermilch aufgenommen worden sein könnten. Es gelang in Milch und Serum der Ammen, sowie im Serum der Jungtiere, T. gondii spezifische Immunglobuline nachzuweisen. Die Transmission des Parasiten als freier Tachyzoit wurde in dieser Arbeit simuliert. Tachyzoiten von T. gondii wurden in verschiedener Dosierung in Rattenmilch angereichert und 48 Stunden alten Ratten verabreicht. Die humorale und zelluläre Immunantwort wurde getestet. Tachyzoiten, die über die Milch aufgenommen wurden, können eine Infektion auslösen. Ratten wurden schließlich auf natürlichem Wege über Milch mit T. gondii infiziert, die humorale Immunantwort bestimmt und der Gehalt der infizierten Rattenmilch an Immunglobulinen überprüft. Die Antikörperkonzentration in Serum und Milch der Ammen zeigte eine deutliche Korrelation und im Serum der Nachkommen ließen sich ebenfalls Antikörper nachweisen. Zeichen einer Infektion fanden sich jedoch nicht. Die Rattenmilch kann also T. gondii und toxoplasmaspezifische Immunglobuline enthalten, die von Nachkommen aufgenommen werden. Den Mechanismus der Parasitenübertragung und die Rolle maternaler parasitenspezifischer Immunglobuline für Infektion und parasitenspezifische Immunantwort der Nachkommen gilt es jedoch noch zu klären.
iNKT cells are a population of T cells with unique characteristics. In contrast to most αβ T cells which recognize peptides presented by highly polymorphic MHC molecules, iNKT cells are reactive to glycolipids presented by CD1d, a non-polymorphic MHC-I like molecule. Moreover, whereas MHC-restricted αβ T cells bear highly variable receptors (TCRs) formed after somatic recombination of the V(D)J gene segments, the TCR of iNKT cells is formed by an invariant α chain, which always contains the same gene segments: AV14 and AJ18; and a β chain of limited BV gene usage: BV8S2, BV7 or BV2, in the mouse. This invariant α chain is the reason for which these cells are named “i” and the NK part of their name refers to the expression of receptors typical of natural killer (NK) cells. iNKT cells recognize glycolipids of endogenous and microbial origin. After activation they secrete large amounts of very different cytokines such as IFN-γ and IL-4 and thus influence immune responses and pathological conditions. One of the most potent iNKT cell agonists, recognized by the semi-invariant TCR, is the synthetic glycolipid α-Galactosylceramide (α-Gal). iNKT cells can be visualized using CD1d-multimeric complexes loaded with α-Gal and flow cytometry, since this reagent has enough avidity to stain these cells. Interestingly, mouse iNKT cells can be stained with human α-Gal-loaded CD1d oligomers and human iNKT cells can also be visualized with mouse α-Gal-loaded CD1d oligomers, indicating a high degree of conservation of the recognition of α-Gal presented by CD1d through evolution. Previous studies showed that rats have the genes necessary to build semi-invariant TCRs: They have a CD1d homologue; one or two BV8S2 homologues and interestingly, up to ten AV14 gene segments, which are highly conserved when compared to the mouse genes. Importantly, it has been shown at least for two of these AV14 gene segments that they can produce invariant TCRα chains which, when coexpressed with BV8-containing β chains, react to α-Gal presented by rat CD1d. Furthermore, ex vivo stimulation of primary splenocytes with α-Gal results in the secretion of IL-4 and IFN-γ. Surprisingly, rat semi-invariant TCRs do not recognize α-Gal presented by mouse CD1d and accordingly, mouse α-Gal-loaded CD1d tetramers failed to stain a discrete population of rat iNKT cells. Taking all together, despite that strong evidence suggested that iNKT cells are present in the rat, the direct identification of such population and the analysis of CD1d-restricted immune responses were still pending for this species. Hence the work presented in this doctoral thesis was aimed to identify iNKT cells, to analyze their phenotype and also to study the distribution and function of CD1d in the rat. For these purposes, we produced essential reagents which were still lacking such as rat specific anti-CD1d monoclonal antibodies and rat CD1d oligomers. Importantly, two of three anti-rat CD1d monoclonal antibodies (all of them generated in our laboratory before this thesis was initiated) also recognized mouse CD1d and therefore allowed a direct comparison of CD1d expression between rat and mouse. Whereas CD1d distribution in the hematopoietic system was found to be extremely similar between these two species; in non-lymphatic tissues important differences were observed. Interestingly, CD1d protein was detected at not yet described sites such as the rat exocrine pancreas and rat and mouse Paneth cells. These monoclonal antibodies did not only allowed the analysis of CD1d expression, but also the first demonstration of the function of rat CD1d as an antigen presenting molecule, since cytokine release in response to α-Gal was blocked when they were added to ex vivo cultures of rat primary cells. Staining of primary rat iNKT cells (possible now with the newly generated rat CD1d oligomers) revealed interesting similarities with human iNKT cells. First, we observed that rat iNKT cells are only a minority among all NKR-P1A/B positive T cells. Human iNKT cells constitute also a very small proportion of NKR-P1A (CD161) expressing T cells, whereas in mice inbred strains which express NKR-P1C (NK1.1), most of NKRP1C expressing T cells are iNKT cells. Second, the majority of rat iNKT cells are either CD4 or DN and only a small proportion expresses CD8β. These findings are similar to humans and different to mice which lack CD8+ iNKT cells. Third, analysis of various inbred rat strains demonstrated different iNKT cell frequencies which correlated with cytokine secretion after α-Gal stimulation of primary cells. In comparison to mice, iNKT cell numbers are markedly reduced in rats. In F344 rats, inbred rat strain which released the highest cytokine amounts after α-Gal stimulation, approximately 0.25% and 0.1% of total liver and spleen lymphocytes, respectively, are iNKT cells. In contrast, in LEW rats iNKT cells were practically absent and neither IL-4 nor IFN-γ were detected after stimulation of primary cells with α-Gal. Once more, these frequencies are very close to those observed in humans. Last, as reported for human peripheral blood cells, rat iNKT cells could be easily expanded in vitro by adding α-Gal to cultures of intrahepatic lymphocytes, whereas the expansion of mouse iNKT cells was not possible using the same protocol. The presence of a multimember AV14 gene segment family in the rat is an intriguing characteristic. These AV14 gene segments are extremely homologous except in the CDR2α region. Based on the amino acid sequence of this region they have been divided into two different types: Type I and II. A specific tissue distribution of the different types was proposed in the first study where the presence of several AV14 gene segments was described. We also analyzed the AV14 gene segment usage in F344 and LEW inbred rat strains. In F344 rats we found no preferential usage of either AV14 gene segment type in the spleen and the liver but type II AV14 gene segments appeared more frequently in the thymus. In contrast, LEW rats show a preferential usage of type I AV14 gene segments in all three compartments analyzed: Thymus, spleen and liver. Taken all together, the usage of newly generated reagents allowed to gain novel insights into CD1d expression in the rat and in the mouse and to directly identify rat iNKT cells for the first time. The phenotypic and functional analysis of rat iNKT cells revealed numerous similarities with human iNKT cells. These are of special interest, since rats serve to investigate several pathological conditions including models for autoimmune diseases. The possibility now to analyze iNKT cells and CD1d-restricted T cell responses in the rat might help to understand the pathogenesis of such diseases. In addition, the uncomplicated in vitro expansion and culture of rat iNKT cells should facilitate the analysis of the immunomoldulatory capacities of these cells.
The expression of T-cell-associated serine proteinase 1 (MTSP-1) in vivo during Leishmania major infection was analyzed in genetically resistant C57BL/6 mice and in genetically susceptible BALB/c mice. Using a monoclonal antibody as well as an RNA probe specific for MTSP-1 to stain tissue sections, we found T cells expressing MTSP-1 in skin lesions and spleens of mice of both strains. In skin lesions, MTSP-1-positive T cells could be detected as early as 3 days after infection. Most importantly, the frequency of T cells expressing MTSP-1 was significantly higher in susceptible BALB/c mice than in resistant C57BL/6 mice. These findings suggest that MTSP-1 is associated with disease-promoting T cells and that it may be an effector molecule involved in the pathogenesis of cutaneous leishmaniasis.