A long-lived IL-2 mutein that selectively activates and expands regulatory T cells as a therapy for autoimmune disease.

Susceptibility to multiple autoimmune diseases is associated with common gene polymorphisms influencing IL-2 signaling and Treg function, making Treg-specific expansion by IL-2 a compelling therapeutic approach to treatment. As an in vivo IL-2 half-life enhancer we used a non-targeted, effector-function-silent human IgG1 as a fusion protein. An IL-2 mutein (N88D) with reduced binding to the intermediate affinity IL-2Rßγ receptor was engineered with a stoichiometry of two IL-2N88D molecules per IgG, i.e. IgG-(IL-2N88D)2. The reduced affinity of IgG-(IL-2N88D)2 for the IL-2Rßγ receptor resulted in a Treg-selective molecule in human whole blood pSTAT5 assays. Treatment of cynomolgus monkeys with single low doses of IgG-(IL-2N88D)2 induced sustained preferential activation of Tregs accompanied by a corresponding 10-14-fold increase in CD4+ and CD8+ CD25+FOXP3+ Tregs; conditions that had no effect on CD4+ or CD8+ memory effector T cells. The expanded cynomolgus Tregs had demethylated FOXP3 and CTLA4 epigenetic signatures characteristic of functionally suppressive cells. Humanized mice had similar selective in vivo responses; IgG-(IL-2N88D)2 increased Tregs while wild-type IgG-IL-2 increased NK cells in addition to Tregs. The expanded human Tregs had demethylated FOXP3 and CTLA4 signatures and were immunosuppressive. These results describe a next-generation immunotherapy using a long-lived and Treg-selective IL-2 that activates and expands functional Tregsin vivo. Patients should benefit from restored immune homeostasis in a personalized fashion to the extent that their autoimmune disease condition dictates opening up the possibility for remissions and cures.

Selective permeabilization of the blood-brain barrier at sites of metastasis.

BACKGROUND: Effective chemotherapeutics for primary systemic tumors have limited access to brain metastases because of the blood-brain barrier (BBB). The aim of this study was to develop a strategy for specifically permeabilizing the BBB at sites of cerebral metastases. METHODS: BALB/c mice were injected intracardially to induce brain metastases. After metastasis induction, either tumor necrosis factor (TNF) or lymphotoxin (LT) was administered intravenously, and 2 to 24 hours later gadolinium- diethylenetriaminepentaacetic acid, horseradish peroxidase, or radiolabeled trastuzumab ((111)In-BnDTPA-Tz) was injected intravenously. BBB permeability was assessed in vivo using gadolinium-enhanced T1-weighted magnetic resonance imaging and confirmed histochemically. Brain uptake of (111)In-BnDTPA-Tz was determined using in vivo single photon emission computed tomography/computed tomography. Endothelial expression of TNF receptors was determined immunohistochemically in both mouse and human brain tissue containing metastases. Group differences were analyzed with one-way analysis of variance followed by post hoc tests, Wilcoxon signed rank test, and Kruskal-Wallis with Dunn's multiple comparison test. All statistical tests were two-sided. RESULTS: Localized expression of TNF receptor 1 (TNFR1) was evident on the vascular endothelium associated with brain metastases. Administration of TNF or LT permeabilized the BBB to exogenous tracers selectively at sites of brain metastasis, with peak effect at 6 hours. Metastasis-specific uptake ratio of (111)In-BnDTPA-Tz was also demonstrated after systemic TNF administration vs control (0.147±0.066 vs 0.001±0.001). Human brain metastases displayed a similar TNF receptor profile compared with the mouse model, with predominantly vascular TNFR1 expression. CONCLUSIONS: These findings describe a new approach to selectively permeabilize the BBB at sites of brain metastases to aid in detection of micrometastases and facilitate tumor-specific access of chemotherapeutic agents. We hypothesize that this permeabilization works primarily though TNFR1 activation and has the potential for clinical translation.

[Clinical pharmacokinetic trial of intravenous injection of recombinant human lymphotoxin-alpha derivative].

BACKGROUND & OBJECTIVE: Animal experiment showed that recombinant human lymphotoxin-alpha derivate (rhLTalpha-Da) could inhibit tumor growth,activate immunity, sensitize tumors to chemotherapy, and has low toxicity in vivo. rhLTalpha-Da won't accumulate after multiple administrations. This study was to investigate pharmacokinetic profile of rhLTalpha-Da in tumor patients to provide reference for phase II clinical trail. METHODS: The dosage of rhLTalpha-Da was 10, 20, and 33 microg x m(-2) x d(-1) according to phase I clinical endurance trial. rhLTalpha-Da was mixed with 100 ml 5% glucose solution, and then infused over 30 min on each of 5 consecutive days. Blood samples and urine samples were collected before and after infusion at different time points. Enzyme-linked immunosorbent assay (ELISA) and fluorescent bead immunoassay (FBI) were used to detect the concentration of rhLTalpha-Da in blood and urine. The main pharmacokinetic parameters were calculated by 3p97 pharmacokinetic program. RESULTS: From Feb. 2003 to Dec. 2003, 19 patients were enrolled. The linear range, specificity, precision, accuracy, and stability of ELISA method were satisfied. The lower limit of quantification (LLOQ) was 39 pg/ml. The linear range, sensitivity, specificity, intra-assay precision, and accuracy of FBI method were satisfied, but coefficient of variation of inter-assay precision was over 20%. rhLTalpha-Da in pharmacokinetics conformed to be a one-compartment open model:it had been eliminated quickly from serum and could not be detected 2 h after the cessation of infusion. The half lives (t1/2) of 33 microg x m(-2) x d(-1) and 20 microg x m(-2) x d(-1) of rhLTalpha-Da were (0.24+/-0.09) h and (0.25+/-0.10) h; the abundant volumes of distribution (Vd) were (35.8+/-1.6) L/m(2) and (43.3+/-26.0) L/m(2); the clearance (CL) were (343.36+/-63.23) ng x m(-2) x h(-1) and (269.60+/-24.52)ng x m(-2) x h(-1); the areas under concentration-time curve (AUC) were (74.6+/-18.4) ng x h x L(-1) and (99.0+/-17.8) ng x h x L(-1), respectively. CONCLUSIONS: The pharmacokinetics of rhLTalpha-Da after infusion is fitted to one compartment model and its elimination is linear. There is no rhLTalpha-Da accumulation after multiple administrations.

[Phase I clinical trial of intravenous recombinant human lymphotoxin-alpha derivative].

BACKGROUND & OBJECTIVE: Intravenous recombinant human lymphotoxin-alpha derivative (rhLTalpha-Da) is a novel biological antitumor reagent developed in China. This study was to evaluate the tolerance of tumor patients to rhLTalpha-Da, confirm its maximum tolerable dose (MTD) in vivo, and to provide recommending dose for phase II trial. METHODS: The dose escalation of rhLTalpha-Da was as follows: 10 microg.(m2.day)-1, 20 microg.(m2.day)-1, and 33 microg.(m2.day)-. Each group contained at least 3 patients. rhLTalpha-Da was solved in 5% GS (100 ml), and intravenously infused over 30 minutes daily for 5 consecutive days. RESULTS: A total of 24 patients were enrolled. Grade I-III chill and fever were the most common adverse events, with the occurrence rate of 79.2% (19/24). Other adverse events observed were dyspnea (3/24), nausea/vomiting (3/24), headache (4/24), fatigue (2/24), hypotension (2/24), and skin discomfort at irradiation region (2/24). No obvious abnormity of liver and renal functions was observed. The dose-limiting toxicities (DLT), which occurred at dose level of 33 microg.(m2.day)-1, were grade III chill, grade III fever, and grade III dyspnea. Although there was no definite efficacy showed in this primary study, initial response to rhLTalpha-Da was seen on a minority of patients with cancers, including malignant melanoma, mycosis fungoides, and renal carcinoma. CONCLUSIONS: The MTD of rhLTalpha-Da is 33 microg.(m2.day)-1. The recommended dose for phase II clinical trial is 20 microg.(m2.day)-1.

[Results of Befnorin clinical trails in healthy volunteers]. / Rezul'taty klinicheskogo ispytaniia preparata benforina na zdorovykh dobrovol'tsakh.

Clinical trails of Befnorin based on the human recombinant TNF-beta elaborated at the Research Design and Technology Institute of Biologically Active Substances, "Vector" State Research Center of Virology and Biotechnology, were carried out on healthy volunteers in compliance with a decision passed by the Committee of Medical and Immunobiological Preparations, Russia's Health Ministry. Single Befnorin doses of 5-10(4) U, 10(5) U, 5-10(5) U, and 10(6) U were administered as intramuscular injections. Clinical, biochemical and immunological parameters were registered for 7 days after a single dose. The drug had an impact on the below immunity indices: Fc-phagocytosis of monocytes, migration index and migration inhibition index. The dose of 10(5) U was proven to be most effective and safe. Supposedly, the drug can be effective in the treatment of herpetic diseases.

[The activation of phagocytosis by recombinant human tumor necrosis factor-beta]. / Aktivatsiia fagotsitoza rekombinantnym chelovecheskim faktorom nekroza opukholi beta.

The functional activity of phagocytic cells of various types was studied in white non-inbred mice by administering recombinant human tumor necrosis beta (rhTNF-beta). It was shown that rhTNF-beta increased phagocytic activity of the peritoneal exudate, spleen and liver macrophages as well as blood polynuclears. Stimulation of neutrophils was demonstrated in earlier times after administration of the preparation as compared to macrophages (3 h and 24 h, respectively). The duration of the macrophage activation effect and its expression depended on the dose of the preparation and were the most notable when rhTNF-beta was administered in doses of 10(3)-10(5) U/20 g. The addition of reopolyglucin, the polysaccharide filler, didn't remove a stimulatory effect of rhTNF-beta on macrophages, but influenced its dynamics. Multiple administration of the preparation didn't cause the phagocytosis stimulation effect.

Changes of abdominal temperature and circulating levels of cortisol and interleukin-6 in response to intra-arterial infusions of tumor necrosis factor-alpha or tumor necrosis factor-beta in guinea pigs.

The sister proteins tumor necrosis factor (TNF)-alpha and TNF-beta share 35% of their amino acid sequence and a number, but not all, of their biological properties. In the present study we infused amounts of 5 microg/kg TNF-alpha, TNF-beta (both preparations with identical bioactivities) or of solvent (0.9% sterile saline) into the circulation of guinea pigs and studied the effects on abdominal temperature, on the induction of endogenous formation of interleukin-6 and on levels of cortisol in plasma as a parameter of the activation of the hypothalamic-pituitary-adrenal axis. Infusion of TNF-alpha and TNF-beta both resulted in identical circulating TNF-like-activities corresponding to an amount of about 7000 pg/ml. TNF-alpha induced a biphasic fever lasting for more than 6 h, while in response to TNF-beta just the shorter first phase of fever (duration: 120 min) was measured. Circulating interleukin-6 (baseline level: 12-20 International Units (I.U.)/ml) and cortisol (baseline level: 70-120 ng/ml) increased about 6-fold during the first phase of the febrile response 60 min after the start of infusion with TNF-alpha or TNF-beta. Thereafter interleukin-6 and cortisol declined again in response to TNF-beta, but further increased after infusion with TNF-alpha to peak values measured 3 h after the start of infusion (interleukin-6: 258 +/- 52 I.U./ml; cortisol: 790 +/- 167 ng/ml). In animals infused with solvent abdominal temperature and interleukin-6 remained at the baseline values, just cortisol increased slightly. The results demonstrate that TNF-alpha is a much stronger inducer of fever and interleukin-6 production or of HPA-axis activation than TNF-beta in so far as all the investigated responses can be measured for prolonged time in response to TNF-alpha.

Functional effects of lymphotoxin on crushed peripheral nerve.

The effect of lymphotoxin (LT) on the functional recovery of crushed peripheral nerves was studied. Using a specially designed compression device, a 5 mm segment of the right sciatic nerve of rats was subjected to a 100 g crush load with a 2 hr duration. The rats in the experimental and control groups received two doses of LT (20 micrograms/kg each) or the same volume of saline, respectively, administered intraperitoneously 24 hr and 1 hr before the procedure. Walking track tests and histologic examinations were performed at intervals up to 56 days after the crush. Motor functional recovery in the LT pretreated group started at day 7 while the crushed limb in the control group remained totally dysfunctional. The sciatic functional index improved faster in the LT group than in the control group during the second week after the crush and reached a significant difference (P < 0.05) at day 18. Subsequently, both groups had a similar evolution. Histologic results paralleled the functional findings. In conclusion, LT can promote motor functional recovery of crushed rat peripheral nerve in the early stage of regeneration.

Analysis of action mechanism of lymphotoxin in prevention of cyclophosphamide-induced diabetes in NOD mice.

Recently we reported that lymphotoxin (LT) administration protected non-obese diabetic (NOD) mice and BB rats from insulin-dependent diabetes mellitus. In this study we analysed the protection mechanism of LT by using cyclophosphamide (CY)-induced autoimmune diabetes in NOD mice. Pre-administration of 500 or 1000 U of LT three times a week between the age of 4 and 11-13 weeks before CY-treatment strongly inhibited CY-induced diabetes. This inhibition was reproduced by LT pre-administration at an earlier age (4 to 7 weeks) but not at a later age (8 to 11 or 10 to 12 wks). LT post-administration (100 U daily or 500 U twice a week) after CY-treatment at 14 weeks of age also strongly inhibited CY-induced diabetes. Spleen cell transfer was carried out using various combinations of donors and recipients. Spleen cell transfer from the non-diabetic mice, which were LT pre-administered between the age of 4 and 13 wks, to CY-treated mice did not significantly inhibit CY-induced diabetes, while transfer of the cells from the similarly treated mice to irradiated recipients did induce diabetes although the onset of diabetes was significantly delayed. Diabetes was not transferred by spleen cells from diabetic mice to LT pre-administered and CY-treated mice. LT administration did not change subpopulations and adhesion molecule expressions of the spleen lymphocyte. Taken together, these results suggest that LT protects NOD mice from CY-induced diabetes by making the mice resistant to autoimmune diabetes and possibly by suppressing anti-islet effector cells, but not by inducing adoptively transferable suppressor cells, although the precise mechanisms still remain to be elucidated.

Recombinant human tumor necrosis factor-beta entrapped in liposomes formed by a modification of the dehydration-rehydration method retains potent cytotoxic activity on L929 cells in vitro.

Recombinant human tumor necrosis factor-beta (rhTNF-beta) may be encapsulated with high efficiency in phosphatidylcholine and distearoylphosphatidylcholine liposomes, with entrapment values of 93.4% and 92.3%, respectively, by first entrapping the substance in multilamellar vesicles using a high solute-to-phospholipid ratio followed by freeze-drying and then rehydration. The entrapped cytokine retains potent cytotoxic activity on L929 cells in vitro, causing 100% cytotoxicity, equal to that of free rhTNF-beta at a concentration of about 5 x 10(-8) g/ml.

Characteristics of TNF alpha- and TNF beta-induced fever in the rabbit.

Febrile responses of rabbits to recombinant human tumor necrosis factors alpha (TNF alpha) and beta (TNF beta) were compared. Intravenous (0.1-30 micrograms/kg, I.V.) and intracerebroventricular (0.01-2.5 micrograms/kg, I.C.V.) injection of TNF alpha and TNF beta both caused monophasic and biphasic fevers depending on the dose. The magnitude of fever induced by I.V. and I.C.V. injection of TNF beta was significantly greater than that induced by TNF alpha. Moreover, the second peak of TNF beta-induced biphasic fever was attained more quickly than that of TNF alpha-induced fever. Indomethacin given subcutaneously inhibited the fevers produced by I.V. and I.C.V. injection of TNF alpha and TNF beta. Fever induced either by intravenously injected TNF beta or by intracerebroventricularly injected TNF beta was significantly inhibited by I.C.V. pre-injection of indomethacin. On the other hand, indomethacin given intracerebroventricularly did not affect the fever induced by I.V. injection of TNF alpha or that induced by I.C.V. injection of TNF alpha. These results suggest that TNF alpha and TNF beta act on the brain sites responsible for the activation of prostaglandin synthesis in somewhat different ways.

Effects of intracarotid and intravenous infusion of human TNF and LT on established intracerebral rat gliomas.

The effects of recombinant human tumor necrosis factor (TNF) and lymphotoxin (LT) were investigated against two different established rat gliomas. Single preestablished intracarotid (ic) or intravenous (iv) doses (1.5-2.0 x 10(6) units) were administered to Wistar rats with intracerebral C6 gliomas and Fischer 344 rats with intracerebral T9 gliomas. Five days after cytokine treatment, animals were sacrificed and tumor size determined by histopathologic techniques. In Wister rats, ic TNF produced a greater reduction in size of C6 tumors than iv TNF. Experiments with Fischer rats showed that both TNF and LT were more effective when administered ic compared to iv. Furthermore, LT induced a greater reduction in tumor size than TNF. Additional studies on the age-related susceptibility of these gliomas revealed early, 8-day tumors were more sensitive to ic LT than advanced, 14-day tumors. No direct toxicity of these cytokines against the tumor cells was detected in vitro indicating their autitumor effect was mediated by alternate mechanisms in vivo. Thus for regionally confined gliomas ic therapy was superior to iv therapy and LT was more effective than TNF. Cytokine treatment was most effective on earlier tumors and there appeared to be differences in efficacy related to the tumor-host combination.

Augmented anti-proliferative effect in combined use of human lymphotoxin with a nitrosourea derivative, ACNU, and the involvement of glutathione redox cycle.

The cytotoxic or cytostatic effect of the combined use of human lymphotoxin (LT) with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU) on L cells or Meth A tumor cells was studied. Simultaneous addition of LT derived from a human lymphoid cell line with ACNU (200 or 500 micrograms/ml) significantly augmented the cytotoxic effect. Similar augmented inhibition was obtained when LT was added to ACNU-treated L cells. The pre-treatment of Meth A tumor cells with ACNU (25 or 50 micrograms/ml) augmented recombinant human LT-mediated cytostasis. However, the addition of glutathione (1.0 mg/ml) to ACNU-treated Meth A tumor cells significantly nullified the augmented anti-proliferative effect of LT (10 U/ml). These results suggest that augmentation of the anti-proliferative effect on tumor cells could be induced through the combined use of LT with ACNU by lowering the intracellular level of glutathione.

The pharmacokinetic pattern of glycosylated human recombinant lymphotoxin (LT) in rats after intravenous administration.

We have examined the pharmacokinetics of glycosylated recombinant human lymphotoxin (LT) after intravenous bolus injection in rats and compared them with those of tumor necrosis factor (TNF) or LT species. The results are as follows. 1) The mean half-life of glycosylated LT in serum increases for each increase in dose, and the distribution volume (V) and total body clearance [(Cl (total)] tend to decrease for increase in dose. On the other hand, the half-life of TNF also increases for increase in dose, but the V tends to increase for increase in dose and Cl (total) does not change. 2) The glycosylated LT distributes to all organs so far tested except brain, and tends to accumulate to kidney more than other tissues at 6 h after the injection. 3) Nonglycosylated LT produced by E. coli and the glycosylated LT species carrying both N-type and mucin-type sugar moieties (25 kDa LT) have shorter half-lives and higher Cl (total)s than 23 kDa LT carrying N-type sugar moieties alone. The 21 kDa LT, the same species as 23 kDa LT except that it lacks 15 amino acid residues at the N-terminus, disappears much faster than 23 kDa LT and shows higher V and Cl (total). Thus, glycosylated LT shows nonlinear pharmacokinetics like TNF, but the deposition is quite different from that of TNF. The high serum concentration of glycosylated LT depends upon the presence of N-type sugar moieties, but not mucin-type sugar moieties. The N-terminal protein chain of LT also correlates with the serum concentration.

Tumor necrosis factor-alpha strongly potentiates interleukin-3 and granulocyte-macrophage colony-stimulating factor-induced proliferation of human CD34+ hematopoietic progenitor cells.

Previous studies have shown that tumor necrosis factors (TNFs) inhibit the proliferative effects of crude or purified colony-stimulating factors (CSFs) on low density human bone marrow cell fractions. In the present study we investigated the effects of TNF alpha on the growth of highly purified CD34+ human hematopoietic progenitor cells (HPC) in response to recombinant CSFs. In short-term liquid cultures (5 to 8 days), TNF alpha strongly potentiates interleukin-3 (IL-3) and granulocyte-macrophage-CSF (GM-CSF)-induced growth of CD34+ HPC, while it has no proliferative effect per se. Within 8 days, the number of viable cells obtained in TNF alpha-supplemented cultures is threefold higher than in cultures carried out with IL-3 or GM-CSF alone. Secondary liquid cultures showed that the potentiating effect of TNF alpha on IL-3-induced proliferation of CD34+ HPC does not result from an IL-3-dependent generation of TNF alpha responsive cells. Limiting dilution analysis indicates that TNF alpha increases both the frequency of IL-3 responding cells and the average size of the IL-3-dependent clones. The potentiating effect of TNF alpha on IL-3- and GM-CSF-dependent growth of CD34+ HPC is also observed in day 7 colony assays. Under these short-term culture conditions, TNF alpha does not appear to accelerate cell maturation as a precursor morphology is retained. Finally, TNF alpha inhibits the relatively weak growth-promoting effect of granulocyte-CSF (G-CSF), which acts on a more committed subpopulation of CD34+ HPC different from that recruited by IL-3 and GM-CSF. TNF beta displays the same modulatory effects as TNF alpha. Thus, TNFs appear to enhance the early stages of myelopoiesis.

Augmented antiproliferative effect of tumor necrosis factor (TNF), lymphotoxin and glycyrrhizin in combined use with diethyldithiocarbamate on Meth A tumor cells in vitro.

Role of oxygen free radicals in inhibition of proliferation of Meth A tumor cells was studied. Diethyldithiocarbamate (DDC), a chelator which inactivates superoxide dismutase, was used to examine the effect of tumor necrosis factor (TNF), lymphotoxin preparation derived from a human lymphoid cell line (c-LT) and glycyrrhizin (GL) on in vitro proliferation of Meth A tumor cells. High degree of antiproliferative effect was observed when DDC was added simultaneously with TNF to target cells. Similar effect was obtained by the addition of GL. However, augmentation of antiproliferative effect was not observed when c-LT was added. However, augmentation of antiproliferative effect was observed when the target cells had been treated with DDC in advance of the addition of TNF, GL or c-LT. Roles of oxygen free radicals in inhibition of tumor cell proliferation were discussed.

Transient inhibition of liver regeneration in mice by transforming growth factor-beta 1 encapsulated in liposomes.

We determined whether transforming growth factor-beta 1 (TGF-beta 1) encapsulated in phosphatidylcholine and phosphatidylserine liposomes could inhibit the proliferative response of mouse liver subsequent to partial hepatectomy. C57BL/6 mice were hepatectomized (60%) or underwent laparotomy (control). Peak mitotic activity occurred 48 hr after hepatectomy, as did incorporation of [125I]IdUrd into dividing cells of the liver. The number of Kupffer cells in the regenerating liver was constant relative to liver size, and the uptake of circulating liposomes correlated with liver size and the number of Kupffer cells. Liposomes containing saline (control) or TGF-beta 1 were injected i.v. into hepatectomized mice 24 and 36 hr after the operation. Liposome-TGF-beta 1, but not control liposomes, significantly inhibited the uptake of [125I]IdUrd by liver cells when measured 48 hr after hepatectomy. However, by 72 hr after hepatectomy, the uptake of [125I]IdUrd in livers of mice treated with liposome-TGF-beta 1 was similar to or exceeded that found for hepatectomized mice injected with control liposomes. We conclude that liposomes containing TGF-beta 1 can postpone the proliferative response of regenerating mouse liver cells and are therefore suitable carriers for the in vivo use of TGF-beta 1.

Pharmacokinetics and tissue distribution of parenterally administered human alpha-lymphotoxin in normal and meth-A tumor-bearing BALB/c mice.

These in vivo studies examine the pharmacokinetics of parenterally administered purified, native human alpha-lymphotoxin (LT) in normal and Meth-A bearing BALB/c mice. We found that the lytic activity of alpha-LT was inactivated within 5 h in the blood of both normal and tumor-bearing mice in vivo. However, LT bioactivity in vitro was not affected by incubation with fresh serum. Radioiodinated LT was rapidly sequestered in the kidneys of both normal and tumor-bearing animals. Systemically administered, radioiodinated LT did not selectively localize in tumor tissues.

Tumor necrosis factor-alpha and tumor necrosis factor-beta (lymphotoxin) stimulate the production of granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and IL-1 in vivo.

A number of cell types have been shown to elaborate hematopoietic growth factors in response to inflammatory mediators in vitro. To determine if this response occurs in vivo, we have administered levels of TNF-alpha and TNF-beta (lymphotoxin) found during an inflammatory reaction to mice. Using Northern blot analysis to detect tissue levels of hematopoietic growth factor-specific transcripts, and specific biologic and immunologic assays to detect the presence of colony-stimulating factors in the serum, we have found that TNF-alpha and TNF-beta induce the transcription and production of granulocyte-macrophage-CSF, macrophage-CSF, and IL-1. These findings provide an in vivo mechanism for the hematopoietic response to inflammation.