Pierre Vassalli

The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection

Granuloma formation in the liver of mice infected with BCG coincides with local TNF synthesis. Injection of rabbit anti-TNF antibody, after 1 or 2 weeks of infection, dramatically interferes with the development of granulomas (both in number and size, large epithelioid cells failing to appear) and subsequent mycobacterial elimination. Furthermore, fully developed BCG granulomas, after 3 weeks of infection, rapidly regress after anti-TNF treatment. Antibody treatment also prevents or suppresses accumulation of TNF mRNA and protein, which resumes after disappearance of the antibody. Peritoneal macrophages exposed to TNF transiently accumulate TNF mRNA, and show an enhanced increase in TNF mRNA in response to gamma interferon. We propose that TNF released from macrophages in the microenvironment of developing granulomas is involved in a process of autoamplification: acting in an autocrine or paracrine way, it enhances its own synthesis and release, thus favoring further macrophage accumulation and differentiation leading to bacterial elimination.

Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B.

This study characterizes the interaction of murine macrophage nuclear proteins with the tumor necrosis factor alpha (TNF-alpha) promoter. Gel retardation and methylation interference assays showed that stimulation of TNF-alpha gene transcription in peritoneal exudate macrophages was accompanied by induction of DNA-binding proteins that recognized with different affinities four elements related to the kappa B consensus motif and a Y-box motif. We suggest that the basal level of TNF-alpha expression in macrophages is due to the binding of a constitutive form of NF-kappa B, present at low levels in nuclei from resting thioglycolate exudate peritoneal macrophages, to some if not all of the kappa B motifs; we postulate that this constitutive form contains only the 50-kilodalton (kDa) DNA-binding protein subunits of NF-kappa B, not the 65-kDa protein subunits (P. Baeuerle and D. Baltimore, Genes Dev. 3:1689-1698, 1989). Agents such as glucocorticoids, which decrease TNF-alpha transcription, diminished the basal level of nuclear NF-kappa B. Stimulation of Stimulation of TNF-alpha transcription in macrophages by lipopolysaccharide, gamma interferon, or cycloheximide led to an increased content of nuclear NF-kappa B. This induced factor represents a different form of NF-kappa B, since it generated protein-DNA complexes of slower mobility; we propose that this induced form of NF-kappa B contains both the 50- and 65-kDa protein subunits, the latter ones being necessary to bind NF-kappa B to its cytoplasmic inhibitor in uninduced cells (Baeuerle and Baltimore, Genes Dev., 1989). In resting cells, this inducible form of NF-kappa B was indeed detectable in the cytosol after deoxycholate treatment. UV cross-linking experiments and gel retardation assays indicated that the inducible form of NF-kappa B is in a higher-order complex with other proteins.

Tumor Necrosis Factor and Disease Severity in Children with Falciparum Malaria

To investigate the role of tumor necrosis factor in Plasmodium falciparum infections, we measured serum concentrations of this cytokine in 65 Malawian children with severe falciparum malaria. Of these children (mean age, 5.3 years), 55 were unconscious and 10 had hypoglycemia at presentation. Although there was considerable overlap, the mean (+/- SEM) initial serum concentration of tumor necrosis factor was significantly higher in the 10 patients who died (709 +/- 312 pg per milliliter) than in the 55 who survived (184 +/- 32 pg per milliliter; P less than 0.02). The mortality rate increased with the concentration of tumor necrosis factor: at a level of less than 100 pg per milliliter, 1 of 24 patients died; at 100 to 500 pg per milliliter, 6 of 34 patients; and at more than 500 pg per milliliter, 3 of 7 patients. High concentrations of tumor necrosis factor were also associated with hypoglycemia (P less than 0.02), hyperparasitemia (P less than 0.002), age under three years (P less than 0.03), and severity of illness as measured by a prognostic index (P less than 0.0005). The highest serum concentrations of tumor necrosis factor were found in patients who died shortly after admission. The concentrations in cerebrospinal fluid were within the normal range in all patients. In serum samples obtained from 38 convalescent patients, the concentration of tumor necrosis factor declined to a mean of 16 +/- 3 pg per milliliter. We conclude that the level of tumor necrosis factor is frequently increased in patients with severe falciparum malaria, particularly in those with cerebral malaria or hypoglycemia. To determine whether it is important in the pathogenesis of the signs and symptoms of the disease requires further study.

An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis

Transgenic mice expressing high levels of the BclxL or Bcl2 proteins in the male germinal cells show a highly abnormal adult spermatogenesis accompanied by sterility. This appears to result from the prevention of an early and massive wave of apoptosis in the testis, which occurs among germinal cells during the first round of spermatogenesis. In contrast, sporadic apoptosis among spermatogonia, which occurs in normal adult testis, is not prevented in adult transgenic mice. The physiological early apoptotic wave in the testis is coincident, in timing and localization, with a temporary high expression of the apoptosis‐promoting protein Bax, which disappears at sexual maturity. The critical role played by the intracellular balance, probably hormonally controlled, of the BclxL and Bax proteins (Bcl2 is apparently not expressed in normal mouse testis) in this early apoptotic wave is shown by the occurrence of a comparable testicular syndrome in mice defective in the bax gene. The apoptotic wave appears necessary for normal mature spermatogenesis to develop, probably because it maintains a critical cell number ratio between some germinal cell stages and Sertoli cells, whose normal functions and differentiation involve an elaborate network of communication.

Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis. A mouse model of progressive pulmonary fibrosis.

The murine TNF-alpha gene was expressed under the control of the human surfactant protein SP-C promoter in transgenic mice. A number of the SP-C TNF-alpha mice died at birth or after a few weeks with very severe lung lesions. Surviving mice transmitted a pulmonary disease to their offspring, the severity and evolution of which was related to the level of TNF-alpha mRNA in the lung; TNF-alpha RNA was detected in alveolar epithelium, presumably in type II epithelial cells. In a longitudinal study of two independent mouse lines, pulmonary pathology, at 1-2 mo of age, consisted of a leukocytic alveolitis with a predominance of T lymphocytes. Leukocyte infiltration was associated with endothelial changes and increased levels of mRNA for the endothelial adhesion molecule VCAM-1. In the following months, alveolar spaces enlarged in association with thickening of the alveolar walls due to an accumulation of desmin-containing fibroblasts, collagen fibers, and lymphocytes. Alveolar surfaces were lined by regenerating type II epithelial cells, and alveolar spaces contained desquamating epithelial cells in places. Platelet trapping in the damaged alveolar capillaries was observed. Pulmonary pathology in the SP-C TNF-alpha mice bears a striking resemblance to human idiopathic pulmonary fibrosis, in which increased expression of TNF-alpha in type II epithelial cells has also been noted. These mice provide a valuable animal model for understanding the pathogenesis of pulmonary fibrosis and exploring possible therapeutic approaches.

Phagocytosis and inflammatory stimuli induce GM-CSF mRNA in macrophages through posttranscriptional regulation

Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is a powerful growth and differentiation factor which acts on hematopoietic progenitor cells and also activates differentiated granulocytes and macrophages. This study shows that mouse peritoneal macrophages can be induced to accumulate GM-CSF mRNA and to release GM-CSF by inflammatory agents (lipopolysaccharide, fetal calf serum, thioglycolate broth); phagocytosis; and adherence in the presence of fibronectin. GM-CSF mRNA accumulation, which is totally prevented by the corticosteroid dexamethasone and by interferon-gamma, is not accompanied by changes in the genes transcriptional level. No interleukin 3 (multi-CSF) mRNA is detectable in induced macrophages. These findings have implications in the understanding of hematopoiesis and of the inflammation and repair process.

Transgenic mice expressing soluble tumor necrosis factor-receptor are protected against bone loss caused by estrogen deficiency.

To evaluate the role of tumor necrosis factor (TNF alpha) in bone loss resulting from estrogen deficiency, the effects of ovariectomy were explored in six-month-old transgenic mice expressing high blood levels of a soluble TNF receptor type I (sTNFR1)-FcIgG3 fusion protein, which neutralizes TNF alpha, and in their nontransgenic littermates used as controls. These transgenic mice were identical to control mice in bone mass (evaluated by bone mineral density and content) and strength. 12 weeks after ovariectomy, the decrease in bone mass and increase in osteocalcin (marker of bone turnover) found in control mice were not observed in transgenic mice, which were not different from sham-operated mice, transgenic or not. This observation suggests a critical role for TNF alpha in the pathogenesis of bone loss induced by estrogen deficiency, a common cause of morbidity in postmenopausal women.

Tumor-necrosis factor and other cytokines in cerebral malaria: experimental and clinical data.

Evidence is presented here that tumor necrosis factor/cachectin (TNF), is of crucial importance in the pathogenesis of cerebral malaria. First, the central lesion of CM, hemorrhagic necrosis of cerebral vessels, corresponds to lesions observed during other pathological conditions associated with high serum TNF levels, such as endotoxemic shock or administration of TNF. Second, in both mouse and human, there is a close correlation between high serum TNF levels and CM. At least in mouse, high TNF levels and CM depend upon T lymphocytes of the CD4+ phenotype. Third, passive immunization against mouse TNF significantly prolongs the survival of P. berghei-infected CBA/Ca mice, and prevents the development of neurologic signs. Treatment with the anti-TNF antibody also prevents hemorrhagic necrosis of brain vessels. Fourth, in the mouse model, a cytokine cascade including at least GM-CSF, IL-3 and IFN-gamma is required for the elevation of TNF level. This cascade appears to involve two components: (a) a quantitative component: increased accumulation of macrophages results from the concomitant release of IL-3 and GM-CSF, and (b) a qualitative component: macrophage number has not only to be raised, but macrophages need to be activated by IFN-gamma. Fifth, metabolic parameters of CM and its main lesion in both mouse and human, i.e. the hemorrhagic necrosis of small brain vessels, correspond to the known properties of TNF.

Small bowel enteropathy: role of intraepithelial lymphocytes and of cytokines (IL-12, IFN-γ, TNF) in the induction of epithelial cell death and renewal

The small bowel mucosa contains within its villus epithelium a large number of intraepithelial lymphocytes (IEL) which upon activation are cytotoxic and release large quantities of IFN‐γ and TNF; these activities are increased by in vitro exposure to IL‐12. Mice injected with IL‐12 develop severe damage of the villus epithelial cells, in form of apoptosis, necrosis and a third distinct form of cell death, characterized ultrastructurally by progressive cell shrinkage. These lesions are accompanied by a compensatory acceleration of the epithelial renewal, a hallmark of epithelial injury. Use of a variety of mutant mice showed that these lesions require the presence of IEL (all populations being involved, thymus‐dependent as well as natural killer‐T cell IEL) and the release of IFN‐γ. The critical role of IFN‐γ may result in part from its capacity to induce on epithelial cells the expression of target molecules involved in the different cytotoxic pathways used by IEL. However, injection of IFN‐γ into mutant mice lacking IEL showed that IFN‐γ can directly induce villus epithelial damage as well. On the other hand, injection of TNF induces fulminant apoptosis of villus epithelial cells, starting at the top of the villi; however TNF is not required for IL‐12‐induced enteropathy, which is unmodified in mutant mice lacking TNF. We propose that, when activated by their cognate ligands and/or IL‐12 produced by cells in the lamina propria, IEL eliminate infected and senescent epithelial cells through a combination of cytotoxicity and of IFN‐γ and TNF release. This insures the rapid epithelial renewal of the villi, which in turn helps maintain the functional integrity of the barrier.

Additive effects of interleukin 1 and tumour necrosis factor-alpha on the accumulation of the three granulocyte and macrophage colony-stimulating factor mRNAs in human endothelial cells.

The control of haematopoietic colony‐stimulating factors (CSF) gene expression by interleukin 1 (IL‐1) and tumour necrosis factor alpha (TNF‐alpha) in cultured endothelial cells was studied by RNA hybridization and nuclear gene transcription. Both IL‐1 and TNF‐alpha induced, with somewhat different kinetics, a slow but marked accumulation of granulocyte‐macrophage (GM)‐ and granulocyte (G)‐CSF mRNAs in endothelial cells; macrophage (M)‐CSF mRNA increased more rapidly but more moderately. Simultaneous treatment with maximally stimulating concentrations of both IL‐1 and TNF‐alpha had an additive effect on the accumulation of the three mRNAs, suggesting that both mediators act via independent pathways. The mechanism of CSF mRNA accumulation in endothelial cells was explored by nuclear run‐on experiments, which showed that both IL‐1 and TNF‐alpha increase GM‐CSF, G‐CSF and M‐CSF gene transcription to varying degrees.