Gary W. Wood

Selective chemokine mRNA expression following brain injury

Injury in non-neuronal tissues stimulates chemokine expression leading to recruitment of inflammatory cells responsible for orchestration of repair processes. The signals involved in directing repair of damage to the brain are less well understood. We hypothesized that following brain injury, chemokines are expressed and regulate the rate and pattern of inflammatory cell accumulation. The two chemokine subfamilies are alpha(alpha)-chemokines, which primarily function as neutrophil chemoattractants, and the beta(beta)-chemokines, which function primarily as monocyte chemoattractants. We assessed alpha and beta chemokine mRNA expression patterns and leukocyte accumulation following a cerebral cortical lesion. Cortical lesions were produced with and without addition of endotoxin, Escherichia coli lipopolysaccharide (LPS), which stimulates cytokine expression. We studied the expression of the beta-chemokines: monocyte chemoattractant protein (gene product JE; MCP-1/JE), macrophage inflammatory protein-1 alpha and beta (MIP-1alpha and MIP-1beta), and the regulated upon activation normal T expressed and secreted chemokine (RANTES) as well as the alpha-chemokines: interferon-gamma-inducible protein (IP-10) and N51/KC (KC; a murine homologue of MIP-2). Changes in gene expression were analyzed by Northern analysis at different time points following injury. Leukocyte and macrophage densities were analyzed by immunohistochemistry at the same time intervals. All chemokines were elevated following cortical injury/endotoxin. MCP-1 and MIP-1alpha were elevated at 2 h and peaked 6 h, MIP-1beta peaked at 6 h, but declined more rapidly than MCP-1 or MIP-1alpha, and IP-10 peaked at 6 h and showed the most rapid decline. KC was elevated at 1 h, and peaked at 6 h following LPS. RANTES was elevated at 1 h and achieved a plateau level between 6 and 18 h, then declined. In contrast, sterile injuries produced in the absence of endotoxin only induced the mRNA of the beta-chemokine MCP-1, and its expression was delayed compared to the cortical injury/endotoxin group. The presence of chemokine message as early as 1 h indicates that expression of this class of molecules is an early response in the repair process following traumatic brain injury. Macrophage/microglia accumulation occurred more rapidly, activated microglia further from the lesion border, and more cells accumulated in cortical injury/endotoxin than in cortical lesions produced under sterile conditions. Thus, there was a positive correlation between beta-chemokine expression and the number of beta-chemokine responsive cells (i.e. microglia) accumulating in injury sites. This is the first comprehensive study using a panel of chemokine probes and specific marcophage/microglial markers to study in vivo activation of the brain following injury. Our data show that the brain is capable of expression of multiple chemokine genes upon appropriate stimulation (e.g. LPS-treatment). The gradient of microglial activation is consistent with physical damage stimulating release of chemokines that diffuse from the injury site. These data strongly suggest that chemokines are instrumental in the initiation of repair processes following brain injury.

Determination of the number and distribution of macrophages, lymphocytes, and granulocytes in the mouse uterus from mating through implantation.

The concentration and distribution of F4‐80 positive cells (macrophages) and common leukocyte antigen (CLA) positive (bone marrow derived) cells were assessed in mouse uterus between days 1 and 8 of pregnancy. High numbers of polymorphonuclear leukocytes and lymphocytes were present on days 1 and 2, but not thereafter. Granulocytes were found both in the endometrium and within the luminal epithelium. The percentage of total cells contributed by macrophages was high on days 1 and 2. That percentage decreased significantly on day 3, then increased again on day 5 and remained high through day 8. Macrophages always were found in myometrial stroma. Macrophages were found throughout the endometrium on days 2 through 8. High numbers of macrophages were observed near epithelia, particularly on days 1,2,4, and 5. Few F4‐80+ or CLA+ cells were observed within the developing primary and secondary decidua. The results demonstrate that an inflammation‐like cellular response occurs in the uterus following mating and that macrophages are a major cellular component of the uterus during early pregnancy.

Interleukin-1, interleukin-6, and tumor necrosis factor α are produced in the mouse uterus during the estrous cycle and are induced by estrogen and progesterone☆

The ovarian steroids, estrogen and progesterone, regulate cellular and molecular changes which occur in the uterus during the estrous cycle. Cycles of protein synthesis, cell proliferation and differentiation, and cell death are the direct results of changes in hormone concentration. To explore the possibility that cytokines, which stimulate proliferation and differentiation of numerous types of cells, might be associated with those cyclic changes, the production of IL-1, IL-6, and TNF alpha was examined in the mouse uterus. Cytokine mRNA expression, bioactivity, and immunoreactivity were quantitated during the estrous cycle, following ovariectomy and exposure of ovariectomized mice to estrogen and progesterone. IL-1, IL-6, and TNF alpha mRNA was detected, and mRNA levels for each of the cytokines varied with the stage of the cycle. Cytokine bioactivity was expressed throughout the cycle, but levels of each cytokine were highest during proestrus and/or estrus. Immunoreactivity paralleled bioactivity. Uterus from ovariectomized mice contained little or no cytokine activity, and systemic administration of estrogen or progesterone resulted in the induction of IL-1 alpha and IL-1 beta mRNA expression. Significant amounts of IL-6 and TNF alpha mRNA appeared only following the exposure of ovariectomized mice to estrogen plus progesterone. Cytokine bioactivity and immunoreactivity also appeared following the administration of estrogen and/or progesterone. The highest activity levels for each cytokine were observed following the injection of estrogen plus progesterone. Cyclic expression of IL-1, IL-6, and TNF alpha in the uterus and their apparent regulation by estrogen and progesterone raise the possibility that cytokines and factors which are induced by cytokines are part of the regulatory process which is induced by ovarian hormones in the uterus of reproductive age females.

Localization and characterization of macrophages in murine uterus.

Macrophages are known to be present in the murine uterus and are known to be among those cells comprising the uterine decidual response to pregnancy. The extent of macrophage involvement in the decidual response has not been documented, and there are unresolved questions regarding expression of markers normally associated with macrophages on cells within the decidua.

Relative role of CSF‐1, MCP‐1/JE, and RANTES in macrophage recruitment during successful pregnancy

It has been previously demonstrated that macrophage colony stimulating factor (CSF‐1) is produced by uterine epithelial cells in response to estrogen and progesterone. Studies in normal and op/op mice demonstrated that accumulation of a portion of the uterine macrophage population could be attributed to the chemotactic properties of CSF‐1. Op/op mice exhibit greatly reduced rates of fertility, but successful pregnancy is not completely blocked. Also, uteri from op/op mice are not completely macrophage deficient. There are two possible explanations for this. One is that not all tissue macrophages are recruited from the bone marrow pool; some may be derived from primitive mesenchyme. Alternatively, tissue macrophages may be recruited from the bone marrow pool through expression of other type I chemokines such as JE, RANTES, MIP‐1α, MIP‐1β, IP‐10, and KC. Both RANTES and JE are expressed at higher levels than CSF‐1 during early pregnancy. The variable expression and relative role of these various chemokines in pregnancy was addressed by measuring mRNA expression during the first 8 days of pregnancy and in a pseudopregnant model. The expression of these various genes relative to macrophage numbers and macrophage distribution will be discussed. The relative role of these various factors in preparing the uterus for blastocyst implantation will be discussed. Mol Reprod Dev 46:62–70, 1997.

Cytokines (IL-1β and TNFα) in relation to biochemical and immunological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats

Abstract Previous studies in different strains of rats and mice have shown that the inhibition of gluconeogenesis as a result of reduced liver phosphoenolpyruvate carboxykinase (PEPCK) activity together with appetite suppression play critical roles in the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Recent immunological studies in rats demonstrated that exposure to low doses of TCDD resulted in an early and enhanced IgG response to immunization with sheep red blood cells (SRBC) and an enhanced delayed-type hypersensitivity (DTH) reaction as well as a positive popliteal lymph node (PLN) response. However, high doses of TCDD suppressed the DTH reaction. This study aimed at examining the involvement of cytokines (IL-1 and TNF) in mediating the above effects. Liver samples from a previous dose-response study on DTH reaction were investigated, in which rats were treated with TCDD (1, 3, 10, 30 and 90 μg/kg) and immunized with an antigen. mRNA levels of IL-1β were elevated begining at the 1 μg/kg (non-lethal) dosage group with a maximum increase of about 5-fold above controls in the 90 μg/kg (lethal) dosage group. mRNA levels of TNFα were also significantly elevated begining at the 30 μg/kg dosage group. These results suggest that at low doses of TCDD, increased IL-1β could be responsible for immune function stimulation, whereas at high doses of TCDD, greatly elevated TNFα and IL-1β levies may exacerbate or mediate acute toxicity including immune suppression and related biochemical effects. A time course study (60 μg TCDD/kg without immunization) revealed that liver mRNA levels of TNFα were significantly elevated starting 24 h, and reaching a maximum 48 h after dosing with TCDD. This change was accompanied by a transient increase of mRNA levels of IL-1β at day 4 after TCDD dosage. Thus, these data demonstrated that TCDD alone (without immunization) can cause transient increases of mRNA levels of TNFα and IL-1β in liver. Results from these experiments suggest that TCDD-induced cytokine changes may play important roles in various effects of TCDD.

Autologous tumor cell vaccination combined with adoptive cellular immunotherapy in patients with Grade III/IV astrocytoma

SummaryBrain tumors are highly resistant to treatment. Their diffuse infiltrative nature and the relative inaccessibility of the brain to blood and lymph are barriers to surgical and cytotoxic treatments alike. Preclinical animal studies demonstrated that intravenously administered tumor antigen-specific T lymphocytes will reject tumors growing in the brain. Specifically activated effector T lymphocytes may be generated by in vivo immunization followed by restimulation of antigen-primed T cells with autologous tumor cells in vitro. In order to apply these findings to humans, feasibility studies of combined active immunization and specific adoptive cellular immunotherapy were performed on fifteen patients with recurrent astrocytoma. The objective was to determine whether; 1) T cells could be grown from peripheral blood of patients immunized with autologous tumor cells, and 2) whether stimulated cells could be safely readministered to patients. Patients were immunized with a combination of their own irradiated tumor cells and Bacillus of Calmette and Guerin. Two weeks later, a mononuclear cell-rich fraction of blood was obtained by leukapheresis. Mononuclear cells were cultured with irradiated autologous tumor cells and interleukin-2. Selective expansion of CD4+ and CD8+ T lymphocytes occurred. Intravenous transfer of stimulated cells to the fifteen patients on twenty-four separate occasions with or without systemic administration of interleukin-2 was tolerated with limited toxicity. The studies established the feasibility of conducting controlled studies of the anti-tumor effects of tumor antigen-specific cellular immunotherapy.

Mononuclear phagocytes in the human placenta

Hofbauer cells have been identified by morphological criteria in human placentae and are considered to be mononuclear phagocytes. An increasing recognition of the morphological heterogeneity of mononuclear phagocytes and of the considerable difficulty in their recognition in histological sections has suggested that they may be considerably more numerous in the placenta than has been appreciated. The use of specific markers for mononuclear phagocytes in the present study facilitated their identification and quantitation in the placenta. Approximately 65 per cent of the cells in cell suspensions from placentae at all stages of gestation exhibited avid Fc receptors and were morphologically indistinguishable from mononuclear phagocytes. Most of these cells also were phagocytic and expressed C3 receptors. Furthermore, when cells expressing alpha-naphthyl butyrate esterase positivity as well as high-affinity receptors for immune complexes were visualized in the stroma of chorionic villi, nearly all stromal cells were positive. The results suggest that Hofbauer cells represent the fully differentiated, morphologically-obvious end-stage members of a large mononuclear phagocyte population which is present in chorionic villi throughout gestation.

The Failure of Microglia in Normal Brain to Exhibit Mononuclear Phagocyte Markers

The origin of brain macrophages or “reactive microglia” has been the subject of considerable controversy. The fundamental question is whether or not there is a morphologically and functionally distinct population of cells, called microglia, which are resident in normal brain and differentiate into macrophages in response to inflammatory stimuli. The present study was performed to determine if any cells in the normal brain have the common markers of mononuclear phagocytes; phagocytosis, IgGFc receptors or macrophage specific antigens. In studies of the newborn and the adult murine brain and adult human brain no cells were detected which had any of those markers, although the highly sensitive marker methods were capable of detecting mononuclear phagocytes in all other tissues where they are known to occur. The results suggest that microglia, if they exist as a distinct cell type, are unrelated to mononuclear phagocytes. Furthermore, they suggest, but do not prove, that all inflammatory macrophages are derived form hematogenous precursors.

Macrophages in murine uterus are immunosuppressive

The mechanisms by which the fetal allograft is protected from a maternal anti-fetal immune response are not understood. This study was designed to examine the possibility that tissues near the developing fetus contain immunoregulatory cells and to begin the process of identification of those cells. Dispersed uterine cell suspensions from pregnant Swiss/Webster mice consistently inhibited the responses of normal murine spleen cells to the polyclonal mitogen phytohemagglutinin (PHA). These suspensions contained few lymphocytes (mean 1%), but abundant macrophages (mean 28%), identified by morphology and Fc gamma-receptor expression. Depletion of Fc gamma-receptor-positive cells restored spleen cell (SC) responses to PHA to near normal levels and partial depletion of adherent cells provided varying degrees of relief of the observed suppression. Adherent cells (greater than 95% macrophages) recovered from plastic surfaces were highly immunosuppressive. Suppressor cells appeared to interfere with both early and late stages of spleen cell proliferative responses. The results suggest that cells with some characteristics of macrophages within tissues near the maternal-fetal interface may create a local environment prohibitive to maternal lymphocyte proliferation.