A reevaluation of B-lymphocyte levels in peripheral blood from cancer patients.

B-lymphocytes were quantitated in mononuclear cell suspensions derived from the peripheral blood of patients with various nonlymphoreticular cancers. The method used was anti-IgM and anti-IgD membrane immunofluorescence. The mean percentage of circulating B-lymphocytes in 78 cancer patients tested was 5.3 +/- 4.6 with a range of 0--18%. Those values were compared with a mean of 9.4 +/- 4.0 and a range of 3--20% for 46 apparently normal individuals. The difference was highly significant (P less than or equal to 0.001). The mean percentage of B-cells in cell suspensions from 43 patients that were tested prior to treatment was 5.8 +/- 4.8 with a range of 0--18%. Very low values were observed both in the presence and absence of therapy, and a correlation with stage of disease could not be established. The low values were associated with decreased T-cell numbers and significantly increased monocyte levels. The fact that those values were significantly lower than have been reported previously for cancer patients was discussed as was the identity of the cells that previously had been counted as B-lymphocytes.

In vitro reversal of depressed T-lymphocyte function in the peripheral blood of brain tumor patients.

Peripheral blood mononuclear cells from some brain tumor patients exhibited depressed T-lymphocyte responses to the polyclonal mitogen phytohemagglutinin (PHA). These responses, initially depressed, were restored to near normal by a 24-hour preculture in growth medium. The effect of preculture was mimicked by mild trypsinization before addition of PHA. In addition, cells treated with deaggregated antihuman IgG resulted in greatly reduced responses to mitogen by mononuclear cells from all brain tumor patients. Anti-IgG had no effect if cells were precultured for 24 hours. The results suggest that, in brain tumor patients, depressed immune function associated with tumor progression was caused by suppressor cells which were activated by humoral factors that express IgG antigenic determinants.

Immunohistologic evaluation of the lymphoreticular infiltrate of human central nervous system tumors.

Forty-five nervous system tumors (9 glioblastomas, 9 meningiomas, 15 assorted primary neural tumors including 3 medulloblastomas, and 12 brain tumors metastatic to the brain were analyzed for their content of lymphocytes, granulocytes, and macrophages. Cell suspensions were prepared by enzymatic digestion; lymphocytes and granulocytes were quantitated by morphology following cytocentrifugation, and macrophages were quantitated by IgG EAC (erythrocyte-antibody-complement) rosette formation. EA (erythrocyte-antibody) adsorption to sections of tumor was employed to determine the distribution of the IgG Fc receptor-positive cells within the tumors and to serve as quality control for selective release of Fc receptor-positive cells by enzyme digestion. The 9 glioblastomas had a mean macrophage content of 41% (range: 5-78%); the 9 meningiomas, 42% (range: 5-80%); the 3 medulloblastomas, 6% (range: 2-15%); and the metastatic tumors, 21% (range: 2-50%). Lymphocyte contents were variable but generally less than 10%. Most tumors contained less than 10% granulocytes. EA adsorption demonstrated that Fc receptor-positive cells were distributed throughout the tumor mass, although different types of patterns were observed. There was an excellent correlation between the percent EAC rosette-positive cells in suspensions and the extent of EA adsorption to the tumor sections. The significance of the study primarily rests in the demonstration that most nervous system tumors contain high numbers of infiltrating host cells, primarily macrophages.

Relationship between monocytosis and T-lmphocyte function in human cancer.

This study was designed to: 1) determine the relative number of monocytes in mononuclear cell suspensions derived from the peripheral blood of cancer patients and 2) ascertain if any relationship existed between the numbers of monocytes in those cell suspensions and T-lymphocyte function. Monocytes were quantitated by morphology verified by phagocytosis of antibody-coated erythrocytes. A significant difference (P less than or equal to 0.01) existed between the number of monocytes in suspensions from normal individuals (26.2 +/- 4.3) and cancer patients (38.0 +/- 13.4). The cancer patients were divided into 2 groups: 1) those who exhibited normal in vitro T-cell responses to phytohemagglutinin and 2) those in whom responses were significantly suppressed. The mean number of monocytes in suspensions from the cancer patient group with normal responses was 29 +/- 9, whereas that from the cancer patients with suppressed responses was 47 +/- 11, a highly significant difference (P less than or equal to 0.01). Therefore, the study demonstrated two things: 1) Mononuclear cell suspensions derived from cancer patients exhibited significant monocytosis relative to those from normal individuals and 2) a strong correlation existed between monocytosis and suppressed T-lymphocyte function in vitro.

Exclusive binding of immunoglobulin to Fc gamma receptors on macrophages in 3-methylcholanthrene-induced murine tumors.

Immunogenic, chemically induced, murine tumors generally are infiltrated with large numbers of Fc gamma receptor (Fc gamma R)-positive cells, most of which are mononuclear phagocytes. This study demonstrates in inbred C3H/HeN mice that such immunogenic tumors contain large amounts of cell-bound immunoglobulin (Ig) and that, within the sensitivity limits of indirect immunofluorescence, the tumor-associated Ig is bound exclusively to host-derived Fc gamma R-positive cells. Depletion of Fc gamma R-positive cells from tumor cell suspensions removed all Ig-bearing cells. Specific competitive elution of the endogenous Ig with heterologous nonimmune IgG established that the endogenous Ig was bound through the Fc gamma R. Tumor-associated Fc gamma R-positive, Ig-bearing cells could be detected with soluble antigen-antibody complexes, and mononuclear phagocytes to which the exogenous complexes were bound expressed Fc gamma R at a level significantly above normal. The role of this macrophage-associated Ig in determining the level of interaction between host cells and the tumor remains to be determined.

Immunoglobulin bound in vivo to Fc receptor-positive cells in human central nervous system tumors.

Fifteen human central nervous system tumors of various histopathologic types were assessed qualitatively and quantitatively by indirect immunofluorescence for the presence of in vivo bound IgG, IgA, and IgM. The tumors were selected to reflect varying degrees of infiltration with Fc receptor-positive macrophages. The major purpose of the study was to determine the relative contribution of immunoglobulin (Ig) bound to tumor cells as compared to Ig bound to the Fc receptor-positive host macrophages. Of the 15 tumors, 1 tumor contained no detectable IgG, IgA, or IgM, 2 tumors contained only IgG and IgA bound in a smooth, homogeneous pattern to the surface of tumor cells, and 8 contained only IgG, IgA, and IgM attached to Fc receptor-positive cells. Four tumors contained significant numbers of tumor cells with cytoplasmic Ig, and two of those tumors also were infiltrated with Fc receptor-positive cells with membrane-associated Ig. Ig was removed from Fc receptor-positive cells but not from tumor cells by prolonged washing of sections of tumor at 37 degrees C, which suggested that Ig was associated with Fc receptors. That possibility was strengthened by the observation that the IgG subclass distribution of the Fc receptor-associated Ig was predominantly IgG1 and IgG3, whereas no predominant subclass existed for IgG bound to tumor cells. Furthermore, the Fc receptor-associated Ig appeared to be in the form of antigen-antibody complexes because it had a granular quality and because IgA and sometimes even IgM were involved in the Fc receptor-bound complexes.

Demonstration of immunoglobulin in tumor and marginal tissues of squamous cell carcinomas of the head and neck.

Immunofluorescence (IF) techniques on cryostatcut sections of tumor tissues demonstrated that immunoglobulin was associated with cells of squamous cell carcinomas of the head and neck. The immunoglobulin was found consistently to be of the IgG class; IgM and IgA were detected also but in only one tumor sample each. The C3 component of complement was also in most tumor tissues. The immunoglobulin could be removed from the tissues by being washed with low pH glycine buffer but not with pH 7 buffer, indicating that the immunoglobulin may be in antibody-antigen complexes. All the tissues obtained from the histologically normal margins of the surgical specimens were also positive for the presence of bound immunoglobulin, whereas head and neck epithelial tissues from tumor-free control patients were negative in the IF assays. Preliminary experiments showed that IgG from patients' diluted serum and the IgG fraction isolated from patients' serum would bind to glycine buffer-eluted tumor tissue.

Suppression of Moloney sarcoma virus immunity following sensitization with attenuated virus.

Murine sarcoma virus (Moloney strain) (MSV-M)-induced tumors are unusual in that they regularly appear less than 2 weeks after virus inoculation, progress for 1 to 2 weeks, and are rejected by normal adult BALB/c mice. Rejectio leaves the animals immune to tumor induction. In the present study, presensitization of normal adult BALB/c mice with attenuated MSV-M resulted in an altered pattern of tumor immunity. Injection of active MSV-M into the presensitized animals resulted in tumor induction and rejection similar to that observed in normal animals, but rejection failed to produce protection against the secondary inoculation with MSV-M. After the second inoculation with active MSV-M, tumors appeared and progressed but ultimately were rejected. Over 80% of the mice died, 25% after the primary challenge and the remainder after the secondary challenge. At death, all mice had histological evidence of leukemia which was the probable cause of death. The animals that died following the secondary challenge also had evidence of disseminated MSV-M. Solid tumor nodules were found in skeletal muscle distant from the original site of inoculation, and active MSV-M was isolated from spleen and lungs. The possibility that the results were produced by specific suppression of MSV-Moloney leukemia virus immunity is discussed.

T-lymphocytes and macrophages in primary murine fibrosarcomas at different stages in their progression.

The relative contribution of lymphocytes, macrophages, and granulocytes to the cell content of primary 3-methyl-cholanthrene-induced murine fibrosarcomas was determined at different stages in their progression by differential cell analysis on enzyme-derived single-cell suspensions. Furthermore, immunohistological analyses were performed on the tumors to detect, quantitate, and determine the distribution of T-lymphocytes and macrophages. The T-lymphocyte content of small tumors was very high, and the T-cells were distributed throughout the tumor mass. As the tumor increased in size, there was a marked decrease in the relative T-cell content; most were located at the tumor periphery. Macrophages were present in significant numers in all tumors and appeared to increase in number as the tumors increased in size. Macrophages were distributed throughout the tumor mass, but generally they were more densely distributed near the tumor periphery. Granulocytes were present in low numbers in all tumors. Yeast phagocytosis was used to assess the functional capacity of the macrophage population. The phagocytic capacity of the macrophages was low in the small tumors, increased significantly as the tumors progressed, but dropped to relatively low levels in large tumors. The results represent a preliminary attempt to characterize the dynamics of host cell infiltration of primary immunogenic tumors.

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

The metrial gland and decidua basalis are uterine structures which, during pregnancy in mice and rats, contain bone marrow derived cells. The current study demonstrates that large numbers of bone marrow derived cells, identified by common leukocyte antigen (CLA) positivity, accumulate in the mesometrial uterus between days 6 and 10 and contribute significantly to the cellularity of both the metrial gland and the decidua basalis. The distribution of F4/80+ cells (macrophages) is similar, suggesting that CLA+ cells in the metrial gland and decidua basalis are macrophages. Disappearance of luminal epithelium in the mesometrial uterus between days 11 and 12 leads to regression of metrial gland and decidua basalis and coincident disappearance of CLA+ and F4/80+ cells. A second population of CLA+ and F4/80+ cells appears in association with the development of new uterine luminal epithelium which surrounds the fetus and placenta during the final week of pregnancy. These very large accumulations of macrophages invariably are related to presence of epithelium.

Relationship between macrophage colony-stimulating factor production by uterine epithelial cells and accumulation and distribution of macrophages in the uterus of pregnant mice.

Estrogen and progesterone induce production of macrophage colony-stimulating factor (CSF-1) by uterine epithelial cells, and CSF-1 is produced in the uterus during pregnancy in mice. CSF-1 is a lineage-specific stimulator of macrophage proliferation, chemotaxis, and function. High concentrations of macrophages accumulate in the uterus during pregnancy. Experiments were conducted to determine whether a relationship exists between intrauterine CSF-1 production and the number and distribution of uterine macrophages during pregnancy in mice. The study demonstrated that on day 1 of pregnancy CSF-1 bioactivity levels were high. The number of macrophages in the uterus was also high on days 1 and 2, and macrophages were concentrated at epithelial surfaces. The decrease in CSF-1 bioactivity seen between days 1 and 2 was followed by a decrease in the macrophage concentration. An increase in CSF-1 bioactivity on day 4 was followed by an increase in the concentration of intrauterine macrophages. During the immediate postimplantation period, macrophages were detected primarily in the myometrium and deep endometrium and CSF-1 bioactivity was undetectable. During the second half of pregnancy, when CSF-1 concentrations were very high, the macrophage concentration was also very high and large numbers of macrophages were detected in association with epithelia. The data confirmed the existence of a direct relationship between intrauterine CSF-1 and macrophage accumulation and suggested that macrophages are attracted to epithelial surfaces by CSF-1.

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.

Endotoxin-induced abortion in mice is mediated by activated fetal macrophages.

Leukocyte numbers and function were assessed in uterus, placenta, and fetus during endotoxin-induced abortion. Tissues initially contained high numbers of macrophages but no granulocytes or lymphocytes. Endotoxin treatment resulted in rapid, transient neutrophil accumulation in uterus and placenta. Moderate increases in macrophage numbers occurred in the uterus but there were no changes in tissue distribution. Myometrial, endothelial, and epithelial cells were unaffected by endotoxin but proliferating, differentiated fibroblasts that made up the primary decidua disappeared. As abortion progressed, the proportional representation of macrophages in placenta and embryo increased until, during the late stages of fetal resorption, they constituted nearly all viable fetal cells. At the same time, overall expression of class II major histocompatibility complex gene products increased in maternal and fetal tissue. Leukocyte distribution and macrophage activation data suggested that endotoxin-induced fetal failure is mediated by products of activated maternal and fetal macrophages acting in concert to destroy actively proliferating maternal and fetal cells.

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. Using tissue immunohistology, macrophages were identified in virgin and pregnant murine uteri. A significant increase in macrophage density was noted during all stages of pregnancy. When uteri from virgin and pregnant mice were enzymatically digested, 10% of uterine cells from virgin and 22% from pregnant mice expressed macrophage markers (binding of rabbit antimouse macrophage serum, Fc gamma receptor expression). Double labeling immunofluorescence demonstrated that the two markers were associated with the same cells. Those results were confirmed in "panning" experiments using a monoclonal antimouse macrophage reagent. In cell suspensions from pregnant murine (C3H/HeN) uteri, 50% of cells exhibiting macrophage markers were I-Ak positive, and macrophages accounted for nearly all I-Ak positive cells in uterine cell suspensions. The results of this study demonstrate that the murine decidual response to pregnancy includes an increase in Fc gamma receptor-bearing macrophages and that a relatively high percentage of those macrophages are Ia positive.

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 from hematogenous precursors.

Detection of interleukin-1, interleukin-6, and tumor necrosis factor-alpha in the uterus during the second half of pregnancy in the mouse.

This study demonstrated interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF alpha) in the mouse uterus during the second half of pregnancy (days 9-18). High concentrations of IL-1 alpha mRNA and bioactive IL-1 were detected between days 12-17. IL-1 bioactivity decreased to very low levels as pregnancy approached parturition. IL-6 mRNA was detected from days 9-18, and IL-6 bioactivity approximately paralleled IL-1 bioactivity. High levels of IL-1 and IL-6 bioactivity, but not IL-1 or IL-6 mRNA, were detected in the placenta between days 12-17. Placental IL-1 and IL-6 also decreased to low levels near parturition. TNF alpha was expressed from days 9-17, and a peak of TNF alpha bioactivity was detected during the period immediately before parturition. TNF alpha mRNA and TNF alpha bioactivity were not detected in the placenta. On day 18, the day of parturition, the concentrations of IL-1, IL-6, and TNF alpha mRNA were very low relative to those on other pregnancy days. Immunocytochemistry with antibodies to IL-1, IL-6, and TNF alpha was used to confirm the presence of all three cytokines in uterine cells throughout the second half of pregnancy. Those data showing the kinetics of cytokine production during fetal development raise the possibility that cytokines and cytokine-induced mediators modulate cellular events during the late stages of pregnancy in the mouse.

Localization of renin in trophoblasts in human chorion laeve at term pregnancy.

It is known that renin is present in fetal membranes, with the highest concentration in the chorion laeve (reflected chorion). The purpose of this study was to identify and localize renin in human chorion laeve. Indirect immunofluorescent analysis, using antiserum against pure human kidney renin, revealed a single layer of cells in the chorion with strongly positive fluorescence. The presence of atrophic villi in this layer together with other morphological evidence indicate that the cells which are positive for renin are cytotrophoblasts. Isolated cells were prepared from the chorion by collagenase digestion, followed by filtration and density gradient centrifugation on Percoll. The isolated cells also showed a positive reaction with the immunofluorescent technique. Control experiments with nonimmune serum did not show fluorescent cells. Biochemical analysis using RIA of angiotensin I generated from sheep substrate indicated that most of the renin activity in the isolated cells was present as inactive renin (activated by trypsin). The presence of renin in trophoblastic cells may be of significance in local cardiovascular regulation, events associated with parturition, or pathophysiological manifestations of trophoblastic disease.

Generation of cellular immune responses against a glioma-associated antigen(s).

The study demonstrated that RT2, a highly malignant anaplastic glioma, expresses antigens that make it susceptible to in vivo adoptive immunotherapy with cytotoxic T lymphocyte-containing immune cell populations. Rats were immunized with irradiated RT2 tumor cells and the adjuvant C. parvum. Lymphocytes from immunized rats were restimulated in vitro with irradiated RT2 tumor cells plus interleukin-2 (IL-2). The cells that proliferated and differentiated in vitro effectively killed RT2, but only low levels of cytotoxicity were observed against other histopathologically related and unrelated, syngeneic and allogeneic target cells. Adoptive transfer of immune cells combined with a 5-day course of systemic IL-2 produced specific regression of brain tumors growing as lung microfoci.

Non-Hodgkin's lymphoma: identification of the monoclonal B lymphocyte component in the presence of polyclonal immunoglobulin.

A high proportion of non-Hodgkin's lymphomas are neoplastic proliferations of B lymphocytes, and, as such, express integral membrane and/or cytoplasmic immunoglobulin (Ig). Because these cellular proliferations are monoclonal, the Ig of all neoplastic lymphocytes will have identical light chain type and idiotype. These tumors sometimes contain significant amounts of polyclonal Ig. In this study we demonstrate that the polyclonal non-B-lymphocyte-associated Ig may be removed by washing tissue at low pH to reveal either neoplastic B lymphocytes or neoplastic "null" lymphocytes. These observations should facilitate the application of immunohistology to the routine diagnosis of lymphoma.

Influence of oestrogen and progesterone on macrophage distribution in the mouse uterus.

Macrophages are constituents of all normal connective tissue including the murine uterus. Macrophages have been identified previously in endometrium and myometrium of pregnant and non-pregnant murine uterus using antibodies against macrophages. In the current study immunohistochemical analysis of murine uterus demonstrated that there were not significant quantitative differences in uterine macrophages between the diestrous, pro-oestrous and oestrous stages. However, distributional changes occurred during the oestrous cycle. Macrophages were evenly distributed throughout uterine tissue during dioestrus, while, during pro-oestrus and oestrus, their concentration was highest in the subepithelial stroma. Because the oestrous cycle is hormonally regulated, we asked whether or not oestrogen and/or progesterone might influence macrophage distribution. Ovariectomy, which eliminates cyclical production of oestrogen and progesterone, resulted in a significant decrease in both the relative and the absolute number of uterine macrophages within 6 days. Injections of progesterone or oestrogen to ovariectomized mice resulted in restoration of uterine macrophage numbers. Injection of oestrogen plus progesterone in a regimen known to prepare the uterus for receptivity for blastocyst implantation increased the number of macrophages to levels which were consistently higher than those seen during oestrus. Moreover, following hormone administration macrophages were more concentrated in the subepithelial stroma, a distributional pattern which was most evident following injection of both hormones. The results suggest that both oestrogen and progesterone promote quantitative and distributional changes in the uterine macrophage population.