Andrea Kruse

Mucosal cytokine expression, cellular markers and adhesion molecules in inflammatory bowel disease

OBJECTIVE To relate proinflammatory cytokines to leukocyte surface markers and adhesion molecules in the same paraffin-embedded biopsy specimen in inflammatory bowel disease (IBD) of varying activity. METHODS Biopsies of seven cases of Crohns disease, seven patients with ulcerative colitis, one case of intestinal infection and six control subjects were studied. We performed in situ hybridization on sections of tissue using probes specific to interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF alpha). Leucocyte markers and adhesion molecules were investigated in subsequent slides of selected specimens by immunohistochemistry. RESULTS Cytokine mRNA was found in large numbers of cells throughout the inflamed intestine but also in some macroscopically unaffected tissue specimens. Transcripts were predominantly located within the lamina propria where immunohistochemistry of parallel sections revealed numerous macrophages and the presence of endothelial adhesion molecules. The expression of the different cytokines was closely related to each other and to histological but not to macroscopic (endoscopic) activity. CONCLUSIONS The synthesis of IL-1beta IL-6 and TNF alpha mRNA is coordinately regulated. Cytokine production is located mostly in the lamina propria at sites that are rich in macrophages and show abundant staining of vascular adhesion molecules. This cascade of immune events is related to inflammatory cell infiltration in both Crohns disease and ulcerative colitis.

Cytokine production after heliumneon laser irradiation in cultures of human peripheral blood mononuclear cells

The effects of laser light on the immune system have not been extensively characterized. Low-power laser sources, such as the helium-neon (He-Ne) laser with a wavelength of 632.8 nm, have been found to produce photobiological effects with evidence of interference with immunological functions. We have investigated the effects of He-Ne laser irradiation on Ficoll-Hypaque-isolated human peripheral blood mononuclear cells (PBMC). Cultured cells were irradiated for various times at two selected intensities and then stimulated with different mitogens. The rate of incorporation of 3H-thymidine into the DNA of stimulated cells decreased with increasing energy density. The levels of interleukin-1 alpha (IL-1 alpha), interleukin-2 (IL-2), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in supernatants of the cultures were determined (irradiated either before or after stimulation). When stimulating cells after irradiation, significantly increased levels of all cytokines were detected after 30 min of irradiation (18.9 J cm-2), whereas after 60 min of irradiation (37.8 J cm-2) cytokine levels were found to be significantly decreased.

Alterations in the Expression of Homing-Associated Molecules at the Maternal/Fetal Interface During the Course of Pregnancy

Abstract One of the most fascinating immunologic questions is how the genetically distinct fetus is able to survive and develop within the mother without provoking an immune rejection response. The pregnant uterus undergoes rapid morphological and functional changes, and these changes may influence the nature of local immune responses at the maternal/fetal interface at different stages of gestation. We hypothesized that specialized mechanisms exist to control access of maternal leukocyte subsets to the decidua and that these mechanisms are modulated during the course of pregnancy. At the critical period of initial placenta development, the maternal/fetal interface displays an unparalleled compartmentalization of microenvironmental domains associated with highly differentiated vessels expressing vascular addressins in nonoverlapping patterns and with recruitment of specialized leukocyte subsets (monocytes, granulated metrial gland cells, and granulocytes) thought to support, modulate, and regulate trophoblast invasion. One of the most striking observations at this time of gestation is the almost complete exclusion of lymphocytes from the maternal/fetal interface. The second half of pregnancy is characterized by a partial loss of microenvironmental specialization and different switches in vascular specificity within the decidua basalis, paralleling dramatic changes in the populations of recruited leukocytes (e.g., a striking influx of lymphocytes, especially T cells). In the term pregnant uterus, the expression of all vascular addressins decreased dramatically; only weakly staining maternal vascular segments remained. These segments may define sites of extremely low residual traffic in the term decidua, which contains remarkably few maternal leukocytes overall. Our results suggest that the maternal/fetal interface represents a situation in which leukocyte trafficking is exquisitely regulated to allow entry of specialized leukocyte subsets that may play a fundamental role in immune regulation during pregnancy.

Evidence of specialized leukocyte-vascular homing interactions at the maternal/fetal interface.

In normal pregnancy, the maternal immune system fails to reject the fetus or the placenta as an allogeneic graft. We hypothesize that specialized mechanisms of leukocyte recruitment might limit access of circulating maternal immune cells to the maternal / fetal interface. During the critical period of initial trophoblast invasion there is an elegantly orchestrated progression of leukocyte homing events in the decidua basalis, associated with highly regulated expression of vascular addressins and segregation of specialized leukocyte subsets into well‐defined decidual microdomains. Neutrophils are limited to the region of necrosis associated with enzymatic digestion at the leading edge of the invading trophoblast, where an almost linear array of maternal blood vessels displays the neutrophil ligand E‐selectin. Cells with the phenotype of monocytes but expressing α4β7 integrin are localized in the blood vessels of the specialized “vascular zone”, which display the unusual combination of P‐selectin (partially associated with platelets) and the α4β7 ligand mucosal vascular addressin‐1 (MAdCAM‐1). Granulated metrial gland cells (α4+β7–, probably α4β1+) constitute a well‐defined cluster positioned in the central decidua basalis around venules prominently expressing the α4β1 ligand VCAM‐1 (but not MAdCAM‐1). T and B lymphocytes are rare. Our results suggest that selective mechanisms for regulating leukocyte access, associated with microdomain specialization within the decidua basalis, may play a fundamental role in immune regulation during the invasive period of placental development.

Role of dendritic cells in the regulation of maternal immune responses to the fetus during mammalian gestation.

Successful mammalian pregnancy relies on the action of sophisticated regulatory mechanisms that allow the fetus (a semi-allograft) to grow and develop in the uterus in spite of being recognized by maternal immune cells. Among several immunocompetent cells present at the maternal fetal interface, dendritic cells (DC) seem to be of particular relevance for pregnancy maintenance given their unique ability to induce both antigen-specific immunity and tolerance. Thus, these cells would be potentially suitable candidates for the regulation of local immune responses within the uterus necessary to meet the difficult task of protecting the mother from infection without compromising fetal survival. Current evidence on decidual DC phenotype and function, and their role in the regulation of the maternal immune system during mouse and human pregnancy are discussed and reviewed herein; highlighting novel DC functions that seem to be of great importance for a successful pregnancy outcome.

Functional involvement of P‐selectin and MAdCAM‐1 in the recruitment of α4β7‐integrin‐expressing monocyte‐like cells to the pregnant mouse uterus

Leukocyte recruitment to the pregnant mouse uterus has been suggested to be associated with highly regulated expression of distinct patterns of vascular adhesion receptors. One of the most striking observations is the combined expression of mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1) and P‐selectin by maternal vessels of the vascular zone during the critical period of initial placenta development. The predominant cell population within these vessels is of the monocyte/macrophage lineage and expresses the mucosal integrin α4β7, which represents the ligand for MAdCAM‐1; neutrophils and lymphocytes are rare. To directly assess the importance of identified adhesion receptors, we undertook long‐term in vivo inhibition studies using monoclonal antibodies to inhibit the contribution of MAdCAM‐1 in leukocyte trafficking to the decidua or to deplete α4β7+ leukocytes. In addition, implantation sites of mouse strains genetically deficient in specific adhesion receptors were investigated. Our results underline the importance of predicted adhesion pathways in the recruitment of monocyte‐like cells, especially those expressing α4β7. Interestingly, maternal/fetal units with inhibited recruitment of α4β7+ leukocytes or the absence of these cells are characterized by reduced size and frequency of uterine NK cells.

DC within the pregnant mouse uterus influence growth and functional properties of uterine NK cells

The vascular addressins mucosal addressin cell adhesion molecule‐1, P‐selectin and ICAM‐1 permit α4β7‐integrin‐expressing DC, especially those of the myeloid lineage (CD11c+CD11b+ DC), to access the pregnant mouse uterus. Injection of blocking monoclonal antibodies against mucosal addressin cell adhesion molecule‐1 in P‐selectin−/− mice or experimental approaches with β7‐integrin−/− or ICAM‐1−/− mice revealed that limited access or absence of CD11c+CD11b+ DC at the maternal/fetal interface negatively affects the frequency, size and functional properties of uterine NK (uNK) cells. Adoptive transfer of DC obtained from WT mice into β7‐integrin−/− mice abrogates these effects and emphasizes the importance of DC in uNK cell differentiation. Interestingly, those implantation sites lacking CD11c+CD11b+ DC are characterized by decreased IL‐15 and IL‐12 mRNA and/or protein levels. Chronic administration of IL‐15 in these mice gives rise to uNK cell numbers and size comparable to those of WT mice, whereas additional injection of IL‐12 positively affects the IFN‐γ expression of uNK cells. Real‐time RT‐PCR and protein arrays performed with isolated uterine DC underline the role of DC as a source of IL‐15 and IL‐12 in the pregnant mouse uterus.

Cytokine production of the human monocytic cell line Mono Mac 6 in comparison to mature monocytes in peripheral blood mononuclear cells.

Mono Mac 6 is a human monocytic cell line with several features of mature blood monocytes such as CD 14 antigen expression, phagocytotic ability, and the functional ability to produce cytokines. This line is often used as an in vitro model to demonstrate the actions of monocytes. In our study, the production of cytokines by Mono Mac 6 cells in response to various stimulants was analyzed and compared to that of mature monocytes in peripheral blood mononuclear cells (PBMC). Interestingly, the Mono Mac 6 cells produced IL-1 alpha/beta, IL-6, and TNF-alpha after induction with the lectin phytohaemagglutinin A (PHA), mainly known as a T cell activator. The amount of cytokine release did not decrease in the presence of polymyxin B (Pmb), an inhibitor of LPS-induced effects. Kinetic studies revealed maximum cytokine levels 24h after stimulation, whereas human PBMC produced higher yields of all cytokines and enhancement was observed up to 48 hours after induction. Stimulation with the superantigen derived from the supernatant of mycoplasma arthritidis (MAS) induced expression of IL-1 beta, IL-6, and TNF-alpha, whereas staphylococcus enterotoxin B (SEB) did not induce any cytokine release. Further experiments analyzed the ability of Mono Mac 6 cells to produce IFN-alpha which is an important characteristic of mature monocytes. The cells were induced either with inactivated Newcastle Disease Virus (NDV), Sendai Virus, or the synthetic stimulus poly I:C IFN-alpha expression was not detected on the transcriptional or the protein level. In addition, no co-expression of IL-1 and IL-6 was observed in response to these stimuli. Since NDV, Sendai Virus, and poly I:C represent strong IFN-alpha inducers in peripheral blood monocytes, these data indicate that Mono Mac 6 cells lack the ability to express IFN-alpha. In conclusion, our findings show that this cell line is a potent cytokine producer, but the capacity to produce IFN is apparently deficient.

Stimulation of whole blood cultures in patients with ankylosing spondylitis by a mitogen derived from Mycoplasma arthritidis (MAS) and other mitogens

In this study we compared cytokine production and cell proliferation of immunocompetent cells derived from patients with ankylosing spondylitis (AS) to those from healthy blood donors using a whole blood assay. To this end, blood cell cultures were stimulated with the superantigens MAS (Mycoplasma arthritidis supernatant) and staphylococcal enterotoxin B (SEB) and the plant lectins phytohaemagglutinin (PHA) and concanavalin A (Con A). The number of white blood cells (WBC) and lymphocyte subsets were also determined. Cell proliferation and levels of interferon-γ (IFN-γ), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were measured after stimulation with the different mitogens. An ELISA test was used to analyse supernatant cytokine levels. Individuals with AS showed significantly lower IFN-γ concentrations and markedly lower cell proliferation rates with all tested mitogens than healthy controls, while there was no significant difference in IL-6 synthesis. IL-Iβ levels were slightly impaired in the patient group, but only blood cell cultures stimulates with MAS showed a statistical significance. Furthermore, there was a significant elevation of leucocytes and lymphocytes in patients with AS resulting in higher numbers of CD4-positive cells, which implies a higher CD4:CD8 cell ratio. CD19- and CD8-positive cells were not significantly distinct compared to healthy controls. This deviation in cytokine levels and cell proliferation points to a suppression of T lymphocytes. A disturbed T-lymphocyte function may play a part in the pathogenesis of AS.

Selectin Platelet Plays a Critical Role in Granulocyte Access to the Pregnant Mouse Uterus under Physiological and Pathological Conditions

Abstract Leukocyte recruitment to the pregnant mouse uterus is associated with highly regulated patterns of expression of vascular adhesion receptors. One striking observation is the localized expression of mucosal vascular addressin cell adhesion molecule (MADCAM1) and selectin, platelet (SELP, formerly P-selectin) by maternal vessels in the vascular zone (VZ) during the first half of pregnancy. From midgestation onwards, endothelial cells lining the maternal vessels of the VZ in addition express vascular cell adhesion molecule-1 (VCAM1). The predominant cell population within these vessels is monocyte-like cells. Granulocytes and low numbers of lymphocytes are also present. Murine fetal trophoblast cells are almost devoid of adhesion molecules, including SELP. In contrast, spontaneous abortions of allogeneic pregnancies are characterized by dramatic upregulation of SELP on maternal VZ vessels and on fetal trophoblast cells. Upregulation of SELP is associated with a dramatic influx of highly activated granulocytes, which infiltrate the vessels and tissue of the VZ and the trophoblast. The majority of the activated granulocytes within the trophoblast undergo nuclear fragmentation, which can be detected by TUNEL staining. To demonstrate that SELP is involved in the recruitment of granulocytes to the pregnant uterus, we undertook long-term in vivo inhibition studies using a monoclonal antibody to inhibit the contribution of SELP to leukocyte trafficking to the decidua. In addition, the pregnant uteri of syngeneic Selp−/− × Selp−/− mice were investigated and compared to the controls. Our results clearly demonstrate the importance of SELP for granulocyte access to the pregnant mouse uterus under physiological and pathological conditions.