TNF-α Immunoreactivity Research Articles

Evolution of cerebral tumor necrosis factor-alpha production during human ischemic stroke.

Tumor necrosis factor-alpha (TNF-alpha) is detected in ischemic brain cells in experimental animal models and is believed to play an important role in apoptosis. However, the natural expression of TNF-alpha during human stroke is not known. We examined TNF-alpha immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) in brain samples of stroke victims (n=16) after variable survival (15 hours to 18 days). Systemic TNF-alpha content from a separate cohort including severe or lethal stroke cases (n=26) was also assayed. Neuronal TNF-alpha was demonstrated from 0.6 to 5.4 days after the onset of stroke symptoms, peaking bilaterally during days 2 and 3. Bilateral glial TNF-alpha immunoreactivity was detected during the acute phase, with the astrocytic TNF-alpha expression dominating in later phases and persisting contralaterally to the infarct in more matured phases (17 to 18 days). Invading inflammatory cells were TNF-alpha immunopositive beginning on the third day. Besides, vascular wall structures showed immunoreactivity sporadically. TNF-alpha levels were mostly nondetectable in peripheral blood. TUNEL labeling and TNF-alpha staining overlapped, although not completely, during the first days. The data support the hypothesis that TNF-alpha may be involved both in the acute propagation of inflammatory processes and cell death and possibly in the more delayed reconstitutive processes of human ischemic stroke.

TNF-alpha expression in painful and nonpainful neuropathies.

To determine whether the cytokine tumor necrosis factor alpha (TNF-alpha) acts as a pain mediator in neuropathic pain in humans. In animal models, inflammatory cytokines such as TNF-alpha have been shown to facilitate neuropathic pain. The expression of TNF-alpha was analyzed immunohistochemically in 20 human nerve biopsy specimens of patients with painful (n = 10) and nonpainful (n = 10) neuropathies. Additionally, serum soluble TNF-alpha receptor I (sTNF-RI) levels were determined in 24 patients with neuropathies, 16 of which were painful and 8 that were painless. Colocalization studies by confocal fluorescence microscopy for S-100 and TNF-alpha showed expression of TNF-alpha in human Schwann cells. Patients with painful neuropathies showed a stronger TNF-alpha immunoreactivity in myelinating Schwann cells relative to the epineurial background staining compared with patients with nonpainful neuropathy (0.949 +/- 0.047 vs 1.010 +/- 0.053, p < 0.05). Although there was no difference in sTNF-RI levels between painful (n = 16) and nonpainful (n = 8) neuropathies (sTNF-RI: 1412 +/- 545 pg/mL vs 1,318 +/- 175 pg/mL), patients with a mechanical allodynia (n = 9) had elevated serum sTNF-RI (1627 +/- 645 pg/mL vs 1233 +/- 192 pg/mL, p < 0.05) compared with patients without allodynia (n = 15). TNF-alpha expression of human Schwann cells may be up-regulated in painful neuropathies. The elevation of sTNF-RI in patients with centrally mediated mechanical allodynia suggests that systemic sTNF-RI levels may influence central pain processing mechanisms.

Differential cellular expression of tumor necrosis factor-α and Type I tumor necrosis factor receptor after transient global forebrain ischemia

We examined the expression of tumor necrosis factor-α (TNF-α) and the Type I tumor necrosis factor receptor, (TNFR1), in relation to c- fos, a known regulator gene of immediate cellular responses, after an extended period of global ischemia. The number of TNF-α mRNA expressing cells peaked in most brain areas after 8 h of reperfusion. Significant increases in TNFR1 mRNA expression were evident in the cortex at 2 and 8 h of reperfusion and after 8 h of reperfusion in the CA3/CA4 region of the hippocampus. Transient neuronal c- fos mRNA expression preceded these responses. TNF-α immunoreactivity was seen in neurons⋙oligodendrocytes=perivascular cells=ependymal cells=vessel wall structures. After ischemia/reperfusion, increased TNF-α immunoreactivity was evident only in oligodendrocytes. TNFR1 immunoreactivity in sham brains manifested in bundles of cellular fibers of variable length and thickness. In post-ischemic brains, immunoreactivity in these cellular processes representing mainly astroglial extensions was suppressed at 2 h but recovered partially by 8 and 24 h of reperfusion. In contradiction, transient ischemia-induced TNFR1 immunoreactivity was observed in somas of large cortical neurons, in activated microglia/macrophages, perivascular and endothelial cells. Taken together, the increase in neuronal TNF-α mRNA appeared not to be followed by substantial translation to protein in the cerebral tissue after an extended period of global ischemia. However, there was increased neuronal TNFR1 immunostaining in conjunction with increased immunostaining for TNF-α in oligoglial elements, which suggests signaling to neurons by enhanced oligoglial TNF-α.

Relation of epidermal growth factor receptor expression to goblet cell hyperplasia in nasal polyps

Background: Because the epidermal growth factor receptor (EGFR) system regulates mucin production in airway epithelium, we hypothesized a role for this system in mucus hypersecretion that occurs in nasal polyposis. Objective: We examined the relationship between goblet cell hyperplasia, EGFR expression, and inflammatory mediators produced by eosinophils and neutrophils in nasal polyp tissues. Methods: Nasal polyp tissue samples from 8 patients and nasal turbinate biopsy specimens from 6 normal control subjects were examined for alcian blue/PAS staining, mucin MUC5AC (MUC5AC), and EGFR immunoreactivity and EGFR gene expression (in situ hybridization). We also examined the role of eosinophils and neutrophils in goblet cell hyperplasia. Results: In control nasal mucosa alcian blue/periodic acid–Schiff– and MUC5AC-stained areas were 18.40% ± 1.31% and 21.89% ± 1.43%, respectively. In polyps the alcian blue/periodic acid–Schiff– and MUC5AC-stained areas were 51.30% ± 5.85% and 52.07% ± 6.58%, which was significantly larger than that found in control subjects (each comparison, P < .01). Four of 6 control specimens expressed EGFR messenger RNA and protein weakly in the epithelium. In polyps 4 of 8 specimens expressed EGFR gene and EGFR protein strongly; the EGFR-stained area was greater in hyperplastic than in pseudostratified epithelium. TNF-α immunoreactivity, expressed in eosinophils, was increased in EGFR-positive polyps compared with EGFR-negative polyps, suggesting a role for TNF-α in EGFR expression. Neutrophils were increased in the epithelium of EGFR-positive compared with EGFR-negative polyps, suggesting a role for these cells in mucin expression and in goblet cell degranulation. Conclusion: These data suggest a role for EGFR cascade in the regulation of goblet cell mucins in nasal polyps. Proof of concept will require clinical studies using selective EGFR inhibitors. (J Allergy Clin Immunol 2000;106:705-12.)

Tumor Necrosis Factor-Alpha in Ileal Mast Cells in Patients with Crohn’s Disease

Background: Reports that both intestinal and extraintestinal Crohn’s disease (CD) had healed successfully after treatment with anti-tumor necrosis factor-alpha (TNF-α) antibody have strengthened the hypothesis that it has a role in the treatment of CD. The macrophage is one source of TNF-α. Intestinal mast cells are also thought to have a role in CD, but it is not known if human ileal mast cells express TNF-α. Aim: To find out whether TNF-α is expressed by mast cells in the ileal wall in CD patients and controls. Methods: TNF-α was sought immunohistochemically in full thickness specimens of ileal wall from patients with CD (histologically normal, n = 9; inflamed, n = 6) and controls (patients with colonic cancer, n = 8). Mast cells were identified by metachromasia and anti-mast cell tryptase immunoreactivity. Results: In all layers of the ileal wall, and in every specimen investigated, mast cells were the main cell type that expressed TNF-α immunoreactivity out of the TNF-α-labelled cells. The number of TNF-α- labelled mast cells was greater in the muscularis propria in patients compared with controls, both in uninflamed (1.7-fold, p < 0.05) and in inflamed bowel (4.6-fold, p < 0.002); greater in the submucosa in inflamed compared with uninflamed CD (1.6-fold, p < 0.01), and less in the lamina propria in inflamed compared with uninflamed CD (0.4-fold, p < 0.05). Conclusion: Mast cells are an important source of TNF-α in all layers of the ileal wall, and the increased density of TNF-α-positive mast cells in the submucosa and muscularis propria may contribute to the tissue changes and symptoms in CD.

Circulating levels of sTNFR and discrepancy between cytotoxicity and immunoreactivity of TNF-α in patients with visceral leishmaniasis

To study the influence of soluble tumour necrosis factor (TNF) receptors (sTNFR) on bioactivity and immunoreactivity of TNF-alpha in patients with visceral leishmaniasis (Kala-azar) and to examine the association between circulating levels of sTNFR type I and type II with clinical manifestations of the disease. Ten patients with Kala-azar were enrolled. Plasma samples for TNF-alpha and sTNFR were obtained on days 0, 7 and 21-28 of antimonial therapy. Bioactivity of TNF-alpha was measured by cytotoxicity to L-929 cells and immunoreactivity by enzyme-linked immunosorbent assay (ELISA). sTNFR-I and sTNFR-II were measured by ELISA. Measured by ELISA, TNF-alpha was detected at baseline in all patients (range from 22.3 to 163 pg/mL) and showed a linear decline over time on therapy (r = -0.49, P = 0.007). In contrast, when measured by cytotoxicity assay, TNF-alpha was detected in only one patient at baseline (193 pg/mL) and in four patients at the end of therapy (38.7, 95, 133 and 232 pg/mL) and there was no linear association between TNF-alpha and duration of therapy (r = -0.18, P = 0.45). sTNFR-I and sTNFR-II were detected in all patients before therapy. There was a strong positive correlation between plasma concentrations of sTNFR-I and sTNFR-II (r = 0.8, P = 0.006). Levels of sTNFR-I and sTNFR-II declined exponentially with time on therapy. We concluded that sTNFR-I and sTNFR-II are related to disease activity in patients with Kala-azar and that these circulating receptors may interfere with the biological activity of TNF-alpha in patients with Kala-azar.

Transmembrane Tumor Necrosis Factor (TNF)-α Inhibits Adipocyte Differentiation by Selectively Activating TNF Receptor 1

Tumor necrosis factor alpha (TNFalpha) is a potent cytokine with multiple biological activities and exists in two forms as follows: a 17-kDa soluble form that is a cleaved product of the 26-kDa transmembrane form (mTNFalpha). It has been suggested that the transmembrane form of TNFalpha is mainly responsible for localized responses via cell-cell contact. Here, we have examined the activities of transmembrane TNFalpha in cultured adipocytes. A non-cleavable transmembrane form of TNFalpha (mTNFDelta1-9K11E) was expressed in several preadipocyte cell lines using retroviral gene transfer. In wild type preadipocytes carrying both TNF receptors, expression of mTNFDelta1-9K11E resulted in inhibition of the differentiation program. The extent of this varied depending on the nature and strength of the adipogenic stimuli. The TNF receptor responsible for this function was determined by expressing mTNFDelta1-9K11E in preadipocyte cell lines lacking either TNF receptor 1 (TNFR1), 2 (TNFR2), or both. In order to confirm the results in the same cellular background, TNF receptors were also reconstituted in the cell lines lacking corresponding receptors. These experiments demonstrated that TNFR1 was necessary and sufficient for mediating mTNFDelta1-9K11E-induced inhibition of adipogenesis and that this action was similar to that of soluble TNFalpha. In conclusion, our results indicate that mTNFDelta1-9K11E is biologically active in cultured adipocytes and can alter the adipogenic program of these cells by selectively activating TNFR1. This may have physiological implications where local TNFalpha actions are thought to be generated at sites such as adipose tissue.

Mast cells are an important cellular source of tumour necrosis factor α in human intestinal tissue

BACKGROUNDSeveral inflammatory disorders of the intestine are characterised by enhanced expression of tumour necrosis factor α (TNF-α). Monocytes and macrophages have been suggested as a major cellular source of TNF-α...

Tumor necrosis factor alpha expression produces increased blood–brain barrier permeability following temporary focal cerebral ischemia in mice

Alteration of blood–brain barrier (BBB) function occurs in both permanent and temporary cerebral ischemia. Studies in vivo and in vitro have shown that tumor necrosis factor-alpha (TNFα) is involved in changes of BBB permeability. However, the relationship between TNFα expression and BBB disruption during reperfusion is unclear. The aim of this study is to find the cell source of TNFα and to determine the relationship between TNFα expression and BBB disruption following temporary focal cerebral ischemia in mice. Adult CD-1 mice received 1 h middle cerebral artery occlusion (MCAO) followed by 2 h, 6 h, 12 h, 24 h, and 48 h of reperfusion. MCAO was achieved using an intraluminal suture technique and reperfusion was performed by the suture withdrawal. Neutralizing monoclonal anti-mouse TNFα antibody was administrated intraventricularly immediately after reperfusion. TNFα expression was determined by double labeling immunohistochemistry. BBB permeability was determined by albumin immunostaining. TNFα immunoreactivity (IR) was observed in the ipsilateral hemisphere from 1 h MCAO with 2 h reperfusion. TNFα positive cells included neurons, astrocytes, and ependymal cells. BBB disruption was detected beginning at 6 h reperfusion but was not present at 2 h of reperfusion. The areas of BBB disruption were significantly enlarged at 12 h reperfusion and plateaued at 24 h to 48 h reperfusion. BBB disruptions were significantly attenuated in the anti-TNFα antibody treated mice ( p<0.05). Our results demonstrate that TNFα IR existed in neurons, astrocytes, and ependymal cells during reperfusion. TNFα IR following temporary focal cerebral ischemia precedes increased BBB permeability. Treatment with TNFα antibody reduces BBB disruption, suggesting TNFα may be an important mediator in altering BBB permeability during reperfusion.

Upregulation of TNF-α and IL-3 expression in lesional and uninvolved skin in different types of urticaria

Background: Although mast cells are known to secrete a broad spectrum of proinflammatory and immunomodulatory cytokines, the role of these molecules in mast cell–dependent cutaneous inflammation is not clear. Objective: We decided to study biopsy specimens from lesional and nonlesional skin of patients with acute, chronic recurrent, delayed pressure, and cold urticaria; from fleeting wheals of prick test reactions to allergens; and from normal skin of nonallergic subjects. Methods: Cryostat sections were stained by immunohistochemistry with antibodies against IL-3, IL-8, TNF-α, and mast cell–specific tryptase. In serial sections with tryptase and each cytokine, reactivity of mast cells was studied as well. Results: Compared with normal skin and prick test reactions, immunoreactivity for TNF-α and IL-3 was significantly increased on endothelial and perivascular cells of the upper dermis in all urticaria lesions. In nonlesional skin comparable upregulation was noted on endothelial cells and for TNF-α on perivascular cells of patients with delayed pressure urticaria. In addition, TNF-α was expressed throughout the epidermis in lesional and nonlesional skin of patients with all types of urticaria, but not in normal control subjects. Sequential biopsy specimens from patients with cold urticaria showed upregulation of TNF-α and IL-3 on endothelial cells 30 minutes after elicitation of lesions with an ice cube. In contrast to these findings, epidermal immunoreactivity, as well as endothelial and perivascular cell expression of IL-8, were only slightly altered in urticaria compared with normal skin. In sequentially stained sections, few tryptase-positive mast cells reacted to TNF-α, few reacted to IL-3 in pressure urticaria only, and practically none stained for IL-8. Conclusion: These findings suggest that the cytokines studied here are involved in the pathology of urticaria, possibly by inducing subthreshold inflammation in endothelial cells of uninvolved skin. (J Allergy Clin Immunol 1999;103:307-14.)

CYTOKINE RELEASE FROM PLACENTAL ENDOTHELIAL CELLS, A PROCESS ASSOCIATED WITH PRETERM LABOUR IN THE ABSENCE OF INTRAUTERINE INFECTION

There is currently a great deal of interest in the role that cytokines may play in the processes mediating preterm as well as normal term labour. In case of preterm delivery a cause–effect relationship between infection, uncontrollable preterm labour, and increased uterine cytokine concentrations is widely accepted, but there is considerable information that increased uterine cytokine release is also a condition in normal term labour and preterm labour not due to infection. Thereby, the exact cellular sources of cytokine production have not yet been identified. In the present study, the authors used immunohistochemical analysis to localize interleukin 1β (IL-1β) interleukin 6 (IL-6) and tumour necrosis factor α (TNF-α) immunoreactivity within trophoblastic villi and fetal membranes. In the absence of chorioamnionitis, uncontrollable preterm labour, and also normal term labour was associated with strong immunoreactivity for IL-1β and IL-6 in the endothelial cells within trophoblastic villi. In contrast, preterm delivery accompanied by histologically confirmed chorioamnionitis, was not associated with increased expression of cytokine antigens within endothelial cells of the fetal vascular system, but strong cytokine activity was found in polymorphonuclear cells infiltrating the amniochorionic membranes. Therefore, the data suggest two well-defined subgroups among patients delivering preterm. Thereby, increased uterine cytokine concentrations may be realized in both groups, but the cellular sources of cytokine production may be different.

Nuclear Degeneration in the Developing Lens and its Regulation by TNFα

DNA fragmentation in lens fibre cell nuclei undergoing programmed degeneration was identified by terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick end labelling (TUNEL). Lens epithelial cells in culture were induced to differentiate into lens fibre-like clumps of cells (lentoids) by insulin and it was shown that the TUNEL method was also an effective means of labelling degenerating nuclei in lentoid cells in lens epithelial cell cultures. Using immuno-fluorescence and confocal microscopy, it was shown that TNFα and TNF receptor (TNFR1, and TNFR2) immunoreactivity was present in sections of chick embryo lenses. TNFα immunoreactivity was associated with the lens epithelium and lens fibres. TNFR1 immunoreactivity was present in lens epithelial cells, cortical lens fibres, and lens fibre cell nuclei, while TNFR2 immunoreactivity had a similar distribution to that of TNFR1, but was not associated with nuclei. Similar patterns of TNFα, TNFR1, and TNFR2 immunoreactivity were observed in lens epithelial cell cultures. When added to lens epithelial cell cultures, TNFα, at concentrations of 50 to 100 ng ml−1, and agonistic antibodies to both TNFR1 and TNFR2 significantly (P<0.05) enhanced the number of degenerating (TUNEL-positive) nuclei. On the other hand, a neutralising antibody to TNFα significantly (P<0.05) reduced the number of TUNEL-positive nuclei. These results demonstrate that TUNEL is an effective means of labelling degenerating lens fibre nuclei during lens fibre and lentoid differentiation, and suggest a potential role for TNFα-like factors and their receptors in the degeneration of lens fibre cell nuclei during lens differentiation. We further suggest that the nuclear degeneration of lens fibre cells is analogous to the nuclear events that occur during apoptosis, and that in lens cells the nuclear degeneration is uncoupled from the plasma membrane events of apoptosis that normally lead to cell death.

Expression of TNF-alpha by human plasma cells in chronic inflammation.

Tumor necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory cytokine and mediator of the inflammatory response. It has been implicated in the pathogenesis of many inflammatory disorders, including rheumatoid arthritis (RA), septic shock, and Crohn's disease. Using a specific anti-human TNF-alpha antibody we detected immunoreactivity for this cytokine in the cytoplasm of inflammatory cells in several chronic inflammatory disorders, including RA, scleritis, and polyarteritis nodosa. These cells were identified predominantly as IgG-expressing plasma cells. Lymph nodes from patients with Hodgkin's lymphoma and breast cancer, but not from control subjects, were also found to contain TNF-alpha-positive plasma cells. Cultured EBV-B lymphocytes and a human plasma cell line (ARH-77) when stimulated with phorbol myristate acetate demonstrated cytoplasmic TNF-alpha immunoreactivity. Western blot analysis of cell membranes and conditioned media from both cell types revealed the presence of the 26-kDa membrane-bound from and the 17-kDa soluble from of TNF-alpha, respectively. TNF-alpha was quantitated by enzyme-linked immunosorbent assay and found to be biologically active as determined by the L929 cytotoxicity assay. This is the first demonstration that plasma cells may be capable of modulating immune and inflammatory responses, not only by antibody production, but also by their secretion of a key inflammatory mediator, TNF-alpha.

Tumor necrosis factor alpha immunoreactivity of rat peritoneal mast cell granules decreases during early secretion induced by compound 48/80: an ultrastructural immunogold morphometric analysis.

We used fast (seconds) and ultrafast (milliseconds) microwave energy-assisted chemical fixation protocols, postembedding immunogold staining, and a morphometric analysis to investigate the early morphological changes and the TNF-alpha immunoreactivity in the cytoplasmic granules of rat peritoneal mast cells that had been stimulated to secrete by exposure to compound 48/80. Exposure to compound 48/80 induced the development of increased numbers of cytoplasmic granules that exhibited decreased electron density; these granules often also appeared swollen. These granule alterations were accompanied by a significantly decreased proportion of granules that were positive for TNF-alpha immunoreactivity. We also calculated the density of TNF-alpha labeling/mu 2 in both dense (unaltered) and altered granules in specimens. TNF-alpha immunoreactivity was present in dense granules (regardless of whether or not the specimens had been stimulated with compound 48/80) and in cells that were fixed with either fast or ultrafast microwave energy. However, altered granules exhibited a decreased density of TNF-alpha label. These findings show that changes in the immunolocalization and/or density of TNF-alpha immunoreactivity occur very rapidly upon stimulation of rat peritoneal mast cells with compound 48/80.

Immunohistochemical expression of tumor necrosis factor α in neonatal leukomalacia

The expression of tumor necrosis factor ot (TNFα) was examined in infants with leukomalacia by means of immunohistochemical methods with an antihuman TNFα monoclonal antibody. We studied 23 patients with neonatal leukomalacia, classified as having “focal,” “widespread,” or “diffuse” disease according to the distribution of the lesions, and 18 age-matched controls. TNFα immunoreactivity was positive in 19 of the 23 (83 %) patients with leukomalacia, and in 7 of the 18 (39%) controls. TNFα was expressed mainly in glial cells in the deep white matter in both groups, and was most abundant around the necrotic foci in the focal group. TNFα immunoreactivity appeared earlier in patients with leukomalacia than in controls, being first detected at 25 and 29 weeks gestation, respectively. Immunofluorescence double-labeling revealed the TNFα-immunoreactive cells were Ricinus communis agglutinin-1 (RCA-1)-positive microglial cells. Thus, our study revealed increasing expression of TNFα in the normally developing brain during the late fetal period, and overproduction of TNFα by microglial cells associated with the pathogenesis of neonatal leukomalacia.

Endotoxin infusion in anesthetized sheep is associated with intrapulmonary sequestration of leukocytes that immunohistochemically express tumor necrosis factor-alpha.

Plasma levels of tumor necrosis factor-alpha (TNF-alpha) peak between 2 and 4 h during a 12-h continuous infusion of endotoxin in awake sheep. We hypothesized that a source of this TNF-alpha is the pool of leukocytes that accumulate in the pulmonary circulation. To test this hypothesis, we physiologically monitored six anesthetized sheep during baseline and 4-h endotoxin infusion periods (10 ng/kg x min). We obtained open-lung biopsies at baseline and at 20 min and 2 and 4 h during the endotoxin infusion period for immunohistochemical localization of TNF-alpha. The plasma concentration of TNF-alpha increased from an average baseline concentration of 0.06 +/- 0.03 ng/ml (mean +/- SD) to a peak of 1.40 +/- 0.28 ng/ml at 2 h of the endotoxin infusion. We observed increased cytoplasmic TNF-alpha immunoreactivity in situ among neutrophils and intravascular mononuclear phagocytes during the endotoxin infusion compared with baseline. Also, the number of immunopositive leukocytes increased in the pulmonary circulation during the continuous infusion of endotoxin. We conclude that TNF-alpha-producing leukocytes accumulate in the pulmonary circulation during endotoxemia. These cells probably contribute to both the rise in the circulating levels of TNF-alpha and the development of acute lung injury.

Tumor necrosis factor-alpha gene expression in mouse oocytes and follicular cells.

Ovarian cells that transcribe and translate the gene for tumor necrosis factor alpha (TNF alpha) were identified in the adult cyclic mouse by using in situ hybridization and immunocytochemistry. TNF alpha mRNA was observed in > 97% and protein was contained in approximately 53% of the oocytes of healthy follicles with two or more layers of granulosa cells, but neither was detectable in oocytes of primordial follicles and follicles with a single layer of granulosa cells. In early atretic follicles, only 13% contained TNF alpha protein and 40% contained TNF alpha mRNA. In late stages of atresia, intense immunoreactive TNF alpha was observed in all of the oocytes, but TNF alpha mRNA was present in only 13%. In approximately 85% of follicles, theca and/or granulosa cells exhibited TNF alpha mRNA hybridization signals. Macrophage-like cells within the interstitium were positive for TNF alpha mRNA and protein. In corpora lutea, luteal cells and macrophage-like cells contained TNF alpha message, while only the latter lineage contained immunoreactive TNF alpha. Hybridization signals and immunoreactivity were more intense in older corpora lutea than in corpora lutea of the present cycle. Northern blot analysis revealed a 2.2-kb TNF alpha mRNA in the ovary that was unchanged relative to 28S rRNA (constitutive RNA) during the cycle. Similarly, TNF alpha hybridization signals and immunoreactivity did not appear to change throughout the cycle. These results indicate that TNF alpha gene transcription in the oocyte coincides with the synthesis of immunoreactive TNF alpha and that these complex biochemical processes occur at distinct steps of follicular development in the mouse.(ABSTRACT TRUNCATED AT 250 WORDS)