Donald P. Braun

Basal and stimulated secretion of cytokines by peritoneal macrophages in women with endometriosis

OBJECTIVE To evaluate basal (constitutive) and stimulated synthesis of tumor necrosis factor alpha (TNF alpha), interleukin (IL)-8, IL-10 by peritoneal macrophages (PM) in women with endometriosis. DESIGN Peritoneal macrophages were cultured in the presence or absence of lipopolysaccharide (LPS) for 24 hours. Peritoneal fluids (PF) and PM supernatants were assayed for cytokines using ELISA. SETTING Institute for the Study and Treatment of Endometriosis and university-based research laboratories. SUBJECTS Fertile controls undergoing tubal ligation (n = 8) and women with endometriosis (n = 17). INTERVENTION Peritoneal fluid samples were obtained at the time of diagnostic laparoscopy (endometriosis group) or laparoscopy for tubal ligation; both were performed in the midluteal phase of the cycle. RESULTS Both basal and LPS stimulated production of TNF alpha, IL-8, and IL-10 by the PM were elevated significantly in women with endometriosis as compared with the fertile controls. CONCLUSIONS This study demonstrated that cytokines TNF alpha, IL-8, and IL-10 are synthesized at greater than normal levels by basal and stimulated PM from women with endometriosis. The levels of TNF alpha and IL-8 correlated with the levels in the PF, suggesting that PM are the principal source of these cytokines in the PF.

Spontaneous Apoptosis of Endometrial Tissue is Impaired in Women with Endometriosis

OBJECTIVE To evaluate spontaneous apoptosis in single-cell suspensions of eutopic and ectopic endometrium from women with endometriosis and in eutopic endometrium from fertile controls without endometriosis. DESIGN Paired specimens of eutopic and ectopic endometrial tissue from patients with endometriosis and eutopic endometrium from controls were assessed for spontaneous apoptosis. SETTING Institute for the Study and Treatment of Endometriosis and university-based research laboratories. PATIENT(S) Fertile controls (n = 10) and women with untreated endometriosis (n = 16). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Spontaneous apoptosis assessed with an ELISA-based cell death detection kit. RESULT(S) Spontaneous apoptosis (monitored by absorbance) of eutopic endometrium from patients with endometriosis and fertile controls was 0.63 +/- 0.1 and 1.43 +/- 0.11, respectively. Among patients with endometriosis, spontaneous apoptosis of ectopic endometrium was 0.26 +/- 0.06. Decreased apoptosis of ectopic versus eutopic endometrium was observed independent of cycle phase. CONCLUSION(S) The susceptibility of endometrial tissue to spontaneous apoptosis is significantly lower in women with endometriosis than in fertile controls. We suggest that decreased susceptibility of endometrial tissue to apoptosis contributes to the etiology or pathogenesis of endometriosis.

Peritoneal fluid-mediated enhancement of eutopic and ectopic endometrial cell proliferation is dependent on tumor necrosis factor-α in women with endometriosis

OBJECTIVE To determine the effect of autologous peritoneal fluid and tumor necrosis factor-alpha (TNF-alpha) on proliferation of endometrial cells from women with endometriosis. DESIGN Endometrial cells from eutopic and ectopic endometrium were cultured in vitro with peritoneal fluids or recombinant TNF-alpha for 72 hours before DNa synthesis determination by 3H-thymidine labeling and liquid scintillation counting. SETTING An institute for the study and treatment of endometriosis and university-based research laboratories. PATIENT(S) Thirty-five women with endometriosis and 17 controls without endometriosis. MAIN OUTCOME MEASURE(S) In vitro incorporation of 3H-thymidine in endometrial cells was examined. RESULT(S) Peritoneal fluid from women with endometriosis enhanced proliferation of autologous and heterologous endometrial cell cultures from women with endometriosis. The soluble TNF-receptor etanercept blocked the ability of peritoneal fluid from women with endometriosis to enhance proliferation of eutopic or ectopic endometrial cells. Recombinant TNF-alpha also enhanced proliferation of eutopic and ectopic endometrial cells from women with endometriosis. In contrast, autologous peritoneal fluid, heterologous peritoneal fluid from women with endometriosis, and recombinant TNF-alpha failed to enhance, and often inhibited, the proliferation of eutopic endometrial cells from controls without endometriosis. CONCLUSION(S) Endometrial cells from women with endometriosis can utilize factors in peritoneal fluids, such as TNF-alpha, to facilitate proliferation in ectopic environments. Endometrial cells from women without endometriosis do not share this ability, suggesting that this abnormality is etiologically related to development of the disease. Therapy with agents that block the effects of TNF-alpha may be warranted.

The development of cytotoxicity in peritoneal macrophages from women with endometriosis

OBJECTIVE To assess the activation status of peritoneal macrophages from women with endometriosis. DESIGN Peritoneal macrophages from patients undergoing laparoscopy were tested for cytotoxic activity against a cultured hepatoma cell line. SETTING Patients were tested at initial laparoscopy or at the completion of therapy. PATIENTS AND PARTICIPANTS Fertile controls (n = 27), infertile controls (n = 20), untreated endometriosis (n = 43), danazol-treated endometriosis (n = 22), and gonadotropin-releasing hormone agonist (GnRH-a)-treated endometriosis (n = 13) were tested. INTERVENTIONS Danazol (800 mg/d) or GnRH-a therapy for 6 months. RESULTS Cytotoxicity was elevated in stage I and II endometriosis (P less than 0.02) and in infertile controls (P less than 0.05) compared with fertile controls. Cytotoxicity in stage III and IV endometriosis was lower (P less than 0.02) than in stage I and II endometriosis. Indomethacin in vitro increased cytotoxicity (P less than 0.05) in stage III and IV endometriosis but not in the other groups tested. Cytotoxicity in danazol or GnRH-a-treated patients was increased (P less than 0.05 or greater) compared with untreated patients with comparable stage of disease. CONCLUSIONS Peritoneal macrophage cytotoxicity in women with endometriosis is affected by (1) the extent of endometriosis, (2) prostaglandin metabolism, and (3) treatment with danazol or GnRH-a.

Monocyte-mediated enhancement of endometrial cell proliferation in women with endometriosis * †

OBJECTIVE To investigate the capacity of monocytes from women with endometriosis to influence endometrial cell proliferation. DESIGN Uterine endometrial cells were cultured in the presence and absence of autologous blood monocytes for 72 hours before assessment of endometrial cell proliferation by thymidine incorporation. SETTING Patients were tested at initial presentation for evaluation of infertility and/or endometriosis. PATIENTS, PARTICIPANTS Fertile controls, n = 17; infertile controls, n = 9; untreated endometriosis, n = 29. INTERVENTIONS None. RESULTS Endometrial cell proliferation was enhanced significantly by blood monocytes in patients with endometriosis but was suppressed significantly by blood monocytes in fertile controls. Endometrial cell proliferation was not affected significantly by blood monocytes in infertile controls analyzed as a group, but a subset of infertile patients also showed enhancement of endometrial cell proliferation by blood monocytes. CONCLUSIONS Blood monocytes from patients with endometriosis and a subset of patients with unexplained infertility enhance autologous endometrial cell proliferation, whereas blood monocytes from fertile patients suppress endometrial cell proliferation. The capacity of monocytes to enhance endometrial cell proliferation appears to require both monocyte-derived factors that stimulate endometrial cell proliferation and endometrial cells capable of responding to those stimulatory factors. If either of these factors is absent, monocytes either suppress or have no effect on endometrial cell proliferation.

Relationship between apoptosis and the number of macrophages in eutopic endometrium from women with and without endometriosis

OBJECTIVE To investigate the relationship between apoptotic cells and macrophages in the eutopic endometrium of women with and without endometriosis. DESIGN Retrospective analysis of archival uterine endometrial biopsy specimens. SETTING Institute for the Study and Treatment of Endometriosis, and university-based pathology and research laboratories. PATIENT(S) Fifty-one women with endometriosis and 24 healthy control subjects without endometriosis. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The number of TUNEL+ (terminal deoxynucleotide transferase [TdT]-mediated deoxyuridine triphospate [dUTP] nick end-labeling-positive) (apoptotic) cells and CD68+ (CD68 positive) (macrophages). RESULT(S) Apoptotic cells and macrophage numbers were positively correlated in the eutopic endometrium of women with and without endometriosis. However, the number of apoptotic cells and the macrophage content in the endometrium of women with endometriosis was significantly reduced compared with that of healthy control subjects without endometriosis. Differences between apoptosis and macrophage numbers between the two populations were observed predominantly during the early proliferative phase of the menstrual cycle. CONCLUSION(S) The reduction in apoptosis described for endometrial cells in women with endometriosis may be related to reduced macrophage trafficking into the eutopic endomtrium during the early-proliferative phase of the menstrual cycle.

Immunoregulatory cell function in peripheral blood leukocytes of patients with intracranial gliomas.

Levels of indomethacin-sensitive, glass-adherent, preculture-sensitive, and lymphocyte-mediated immunoregulatory activity were measured in peripheral blood mononuclear cells from 12 patients with intracranial astrocytomas. The levels of regulatory cell function were determined in assays of T cell immunocompetence as judged by phytohemagglutinin (PHA)-induced in vitro lymphocyte DNA synthesis. Of 6 patients studied before surgical exploration and diagnosis, 6 exhibited significantly depressed levels of PHA responsiveness in association with significantly increased levels of regulatory cell function by glass-adherent, preculture-sensitive and/or indomethacin-sensitive cells. Six patients were studied after diagnosis and treatment. Of those, 2 in remission had normal levels of immune function in association with normal levels of regulatory cell activity, whereas 2 of 4 patients who had recurrent disease had significantly depressed T cell function in association with increased glass-adherent, preculture-sensitive and/or indomethacin-sensitive regulatory cell activity. Although some alterations in lymphocyte-medicated suppressor cell activity were seen in 2 patients, those changes could not be correlated with impaired T cell function as measured in the PHA stimulation assay. The changes in regulatory cell function could not be correlated with levels of immune complexes in patient sera. These data suggest that increased levels of regulatory cell function by glass-adherent, preculture-sensitive and/or indomethacin-sensitive cells alone or in conjunction with lymphocyte and T cell depletion are a primary determinant of impaired immunocompetence in glioma patients.

Modulation of tumor-infiltrating lymphocyte cytolytic activity against human non-small cell lung cancer

The cytokines expressed in tumor microenvironments are thought to be important mediators of both the host immune response and tumor survival. The source of these cytokines includes tumor cells, infiltrating leukocytes, fibroblasts, and other stromal elements. We previously reported that tumor-infiltrating lymphocytes (TIL) from human non-small cell lung cancer (NSCLC) express predominantly type 1 cytokines, which are known to enhance cell-mediated immunity. The purpose of this study is to assess the cytokine mRNA expression of human NSCLC primary cell lines and the capacity of the tumor-associated cytokines to modulate the development of TIL cytolytic activity against the autologous tumor. Cytokine mRNA expression was determined by RT-PCR and the capacity of TIL to kill autologous lung tumor cells was measured by the chromium-51 (51Cr) release assay. All NSCLC primary cell lines expressed mRNA for IL-4, IL-6, and transforming growth factor-beta1 (TGFbeta1), whereas IL-10 was expressed in only 1/7 cell lines. When added to TIL cultures stimulated with anti-CD3+IL-2, IL-4 and IL-10 enhanced and TGF-beta1 suppressed the development of TIL cytolytic activity against autologous tumor cells. The effects of IL-6 were inconsistent and for the group, were not statistically significant. These results demonstrate that human NSCLC cells express cytokines with the capacity to regulate the in situ anti-tumor immune response. However, the effects of tumor-derived cytokines varied qualitatively and quantitatively suggesting the balance between specific type 2 cytokines or TGF-beta1 within tumor microenvironments may influence prognosis or response to immunotherapy.

A phase I clinical trial of recombinant interleukin-2 by periodic 24-hour intravenous infusions.

Recombinant interleukin-2 (rIL-2) (NSC# 600664; Hoffmann-La Roche, Inc., Nutley, NJ) was studied in a phase I clinical trial in 33 patients with advanced, measureable cancer of the colon or malignant melanoma, Eastern Cooperative Oncology Group (ECOG) performance status O-1, and no prior chemotherapy or radiotherapy. The goal of the study was to identify a dose and schedule of IL-2 to generate maximal immune modulation with tolerable toxicity. Such a regimen might allow the addition of other treatment modalities and/or prolonged treatment duration in later trials. Each patient received IL-2 as a continuous 24-hour infusion once weekly for 4 weeks and then twice weekly for 4 weeks. Five treatment groups received from 10(3) U/m2 to 3 x 10(7) U/m2 per 24-hour infusion. The maximal tolerated dose was 3 x 10(7) U/m2/d twice weekly. Patients treated twice weekly at 1 x 10(7) and 3 x 10(7) U/m2/d had immune modulation in terms of lymphocytosis, eosinophilia, increased natural killer (NK) activity, and elevated numbers of peripheral blood mononuclear cells expressing CD16, OKT10/Leu-17, and Leu-19 surface markers. Endogenous generation of peripheral blood lymphokine-activated killer (LAK) activity was demonstrated by lysis of NK-resistant Daudi targets, in patients treated at 3 x 10(7) U/m2/d. Biochemical and hematological abnormalities were moderate and reversible. Clinical toxicity included hypotension, myalgia, arthralgia, stomatitis, fever, fatigue, nausea, headache, chills, diarrhea, and oliguria at high doses. Cardiovascular toxicity was tolerable for most patients and reversed after IL-2 was stopped. Two of six melanoma patients at 3 x 10(7) U/m2/d achieved partial responses by the end of the eighth week. This IL-2 schedule appears to produce potentially clinically useful immune enhancement with tolerable toxicity.

Impaired tumoricidal function of alveolar macrophages from patients with non‐small cell lung cancer

The capacity of alveolar macrophages and peripheral blood monocytes from patients with non‐small cell lung cancer to develop tumoricidal function after in vitro stimulation with different macrophage activators was investigated. Alveolar macrophages were found to be impaired in their ability to develop cytotoxic activity compared with either the peripheral blood monocytes from the same patients or alveolar macrophages from patients with nonmalignant lung disorders. This result was observed consistently under diverse culture conditions and with different macrophage activators including gamma‐interferon (γ‐IFN), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), phorbol myristate acetate, or endotoxin. The impairment in tumoricidal function observed in alveolar macrophages was not associated with reduced target cell binding compared to peripheral blood monocytes. Alveolar macrophages from patients with lung cancer were found to secrete significantly greater amounts of tumor necrosis factor (TNF) and interleukin‐1 (IL‐1) than either peripheral blood monocytes from the same patients or alveolar macrophages from the patients with nonmalignant disorders. These results are consistent with either different regulatory pathways for cytotoxicity and cytokine secretion in the alveolar macrophages of patients with lung cancer or diversity in the subpopulations of cells responsible for these functions.