Clin. exp. Immunol. (1990) 80, 156-160

Alveolar macrophages in AIDS patients: increased spontaneous tumour necrosis factor-alpha production in Pneumocystis carinii pneumonia

*

V. L. KRISHNAN, A. MEAGERt, D. M. MITCHELL* & A. J. PINCHING Departments of Immunology and Respiratory Medicine, St Mary's Hospital Medical School, London, and t Division of Immunobiology, National Institute for Biological Standards Control,. Potters Bar, Hertfordshire, UK

(Acceptedfor publication 17 November 1989)

SUMMARY and their products in the control of Pneumocystis macrophages In order to assess the role of alveolar carinii pneumonia (PCP) and other infections in AIDS, bronchoalveolar lavage cells and peripheral blood mononuclear cells from .HIV-positive AIDS/ARC patients (with and without PCP) and HIVnegative patients were counted and cultured in vitro; spontaneous and LPS-induced tumour necrosis factor-alpha (TNF-a) production was measured. Markedly increased spontaneous TNF-ca production by alveolar macrophages and, to a lesser extent, peripheral blood monocytes was found in HIVpositive patients with active PCP but not in patients without the infection. Higher TNF production was associated with lower counts of Pneumocystis in the bronchoalveolar lavage fluid. These results suggest that TNF-cx production by macrophages may play an important role in the control of Pn. carinii infection in AIDS. Keywords AIDS factor-alpha

Pneumocystis cariniii pneumonia

INTRODUCTION AIDS is associated with multiple immunological abnormalities, notably of CD4 lymphocyte numbers and function, as a result of HIV infection. Altered function of monocytes and macrophages, which are also targets for HIV, is also important in the pathogenesis of opportunist infections. Several studies have examined monocytes from the peripheral blood of AIDS patients and have characterized functional defects such as impaired chemotaxis, depressed phagocytic and microbicidal activities, poor accessory function and defective interleukin(IL-I) production (Pinching et al., 1983; Smith et al., 1984; Prince et al., 1985; Eales, Moshtael & Pinching, 1987). However, very little is known about the functional status of macrophages in the lung, which is a major target organ for opportunist infections and tumours in AIDS patients. The most common pneumonia is that caused by Pneumocystis carinii (PCP); however, mycobacterial, viral or fungal infections are also often seen. Resident macrophages in the lungs and their products have a central role in controlling pulmonary infection. The increased susceptibility of AIDS patients to such infections may be attributed in part to impaired alveolar macrophage Correspondence: Dr V. L. Krishnan, Institute of Obstetrics and Gynaecology, Hammersmith Hospital, Du Cane Road, London W12 OHS, UK.

alveolar macrophage

tumour necrosis

function, due to either a direct effect of HIV or to lack of activation by lymphokines. Murray et al. (1985) demonstrated that alveolar macrophages from AIDS patients did not have any intrinsic defect in the intracellular killing of Toxoplasma gondii or in their capacity to respond to interferon-gamma (IFN-y). The positive role of macrophages in the control of PCP has hardly been studied. Several macrophage functions are mediated through one of its products, tumour necrosis factor-alpha (TNF-x). Tumour necrosis factor was originally described for its ability to cause necrosis of tumours and also to mediate endotoxin-induced cachexia and lethal shock in Gram-negative infections (Carswell et al., 1975; Beutler, 1985; Caput Beutler & Hartog, 1986). It was later discovered to have several other biological effects and it is now considered to play an important role in the regulation of the immune response and in inflammatory reactions. TNF-cz activates macrophages to express enhanced tumouricidal activity, stimulates IL-I release from macrophages and endothelial cells, stimulates fibroblast proliferation and enhances T cell proliferation (Bachwich et al., 1986; Philip & Epstein, 1986; Vilcek, Palombella & Destefano, 1986; Scheurich et al., 1987). Previous studies of TNF-oc production by peripheral blood monocytes from AIDS patients have given conflicting results. Amman et al., (1987) reported a deficiency in the production of TNF-cx by monocytes, whereas Roux-Lombard et al. (1987), found increased spontaneous and lipopolysaccharide (LPS)

156

Alveolar macrophages in AIDS induced TNF release by these cells. To our' knowledge, TNF-ca production by alveolar macrophages from AIDS patients has not been investigated previously. In this study we have assessed TNF-a release from bronchoalveolar lavage macrophag6s from AIDS patients. Reduced production could contribute to a defective cellular response, while overproduction could enhance local tissue damage. PATIENTS AND METHODS Patient groups included in the study Group A. HIV-positive patients with PCP (AIDS), n=31. Only two patients in this group had other bacterial pneumonias in addition to PCP. Group B1. HIV-positive patients without PCP but with other infections (AIDS and ARC), n = 5. These included streptococcal, staphylococcal, pneumococcal, Haemophilus and Mycoplasma infections. Group B2. HIV-positive patients with negative bronchoscopy, n = 11 (seven with AIDS, four with ARC). Group C. HIV-negative patients who underwent bronchoscopy for haemoptysis or other symptoms and signs suggesting possible malignancy, n = 17. One patient had adenocarcinoma and another had oat cell carcinoma. The remaining patients had negative bronchoscopy. Bronchoalveolar lavage (BAL) was ferformed using 60-ml aliquots of sterile saline which were instilled and aspirated from the right middle lobe during routine diagnostic bronchoscopy. The fluid was filtered through sterile gauze, centrifuged and the cells were washed and resuspended in RPMI medium (Flow) containing 4 mM L-glutamine, 100 U/ml penicillin and 100 pg/ml streptomycin (Glaxo). They were seeded onto 96-well plates at 125 x 105 cells/well; I pg/ml of LPS lipopolysaccharide (Sigma) was added to the wells and the supernatants from these and unstimulated wells were collected after 24 h and tested for TNF-a in triplicates. Viability of the cells maintained in culture was assessed in preliminary experiments by trypan blue exclusion. There was no significant difference between the samples from different patient groups. In subsequent experiments the amounts of TNF released as a result of stimulation ofcells with LPS was used as a check to detect any drastic fall in viability.

Differential counts Cytospin preparations of cells were dried, fixed and stained with May-Grunwald and Giemsa and differential counts were performed based on cell morphology. Immunoperoxidase staining Cytospin preparations were air dried and fixed in cold acetone for 10 min and washed in phosphate-buffered saline (PBS). Fifty microlitres of normal rabbit serum (diluted 1:10 in PBS) were added to the spot, incubated for 20 min at room temperature and washed in PBS; 50 pl of appropriate dilutions of monoclonals were added and incubated for 30 min at room temperature. They were washed again and 50 p1 of 1:100 dilution of peroxidase-conjugated rabbit anti-mouse immunoglobulin (Dakopatts) were added and incubated for 30 min. After washing, slides were incubated in substrate solution containing 0 25% diaminobenzidine hydrochloride and 0 03% hydrogen

157

peroxide in PBS for 10 min. They were then washed in distilled Mayer's haematoxylin.

water and counterstained with

Monoclonal antibodies T4 (anti-CD4), T8 (anti-CD8) (Coulter Electronics); anti-Leu4 (anti-CD3) (Becton-Dickinson); 44 (anti-CD1ib, specific for CR3 receptors on monocytes but not those on tissue macrophages) and 52 (anti-DR) (Courtesy of Dr Nancy Hogg, ICRF,

UK). Enrichment of macrophages Approximately 4 x 106 cells were layered on 4 ml of Nycodenz gradient (Nycomed) and centrifuged at 600 g for 15 min. More than half of the resulting wide band was removed, washed and resuspended in medium. This method gave 85-95% pure macrophages from original samples containing as few as 25% macrophages. But the yield was very low, with only 30-40% recovery and therefore this could only be done with bronchoalveolar lavage (BAL) samples with a high cell count.

Separation of peripheral blood mononuclear cells These (PBMC) were separated from blood on Lymphoprep gradient (Nycomed), washed, resuspended in medium and seeded into wells at 2 5 x 105/well. Supernatants from unstimulated and LPS-stimulated wells were collected after 24 h for TNF-a testing. ELISA for TNF-a The method used was a slight modification of that reported by Meager et al. (1987). Plates were coated with polyclonal antiTNF antibody diluted in coating buffer. The substrate for peroxidase was prepared as follows: O-phenylenediamine dihydrochloride was dissolved in 10% methanol (100 mg in 10 ml). A l-mg/ml solution was made in citrate buffer to which 4pl of 30% hydrogen peroxide were added. After incubation at 37 C for 30 min the reaction was stopped with 1 M sulphuric acid and the plates were read at 492 nm. The limit of detection of this assay was 15 U/ml of TNF. The cut-off point calculated as the 95% confidence limit (mean+1 96xs.e.m.) was 25 U/ml. Values lower than this limit were assigned a value of 25 U/ml.

Grocott staining for Pneumocystis carinii Five millilitres of BAL fluid were centrifuged and the pellet was smeared onto slides and stained by the Grocott method '(-Bancroft 1975). RESULTS

Differential cell counts in BAL fluid There was a marked increase in the percentage of CD8 + T cells in the BAL fluid samples from HIV+ patients as compared with those from HIV- control patients (Table 1). This finding is consistent with previous reports (Wallace et al., 1984). Further, we found that the percentage of CD8 + T cells in the BAL of PCP patients was significantly higher than in the BAL of patients without PCP (significant at I %), suggesting that these cells may play a role in Pneumocystis infection. A marked infiltration of blood-derived monocytes into the lung parenchyma in patients with PCP, was indicated by the presence of large numbers of cells expressing CR3 receptors specific for monocytes in the BAL samples (significant at 1%) (Table 1).

V. L. Krishnan et al.

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Table 1. Cells recovered from BAL, cell counts, and presence of blood-derived monocytes in BAL and DR expression on mature alveolar macrophages Cells in BAL fluid (mean °, + s.e.m.)

Group n A

30

Bl

5

B2

10

C

17

Lymphocytes Cells /ml Blood-derived BAL fluid (mean+s.e.m.) Macrophages Polymorphs CD3Y CD4+ CD8+ monocytes* DR+ cells* 3 25x 105 (+5 3 x 104) 44x 105 (+1 7x 104) 3-2x 105 (+66x 104) 22x 105 (+ 13 x 104)

52+4

9+2

39+4

8+3

31 +2

33+6

60+8

7+4

4+2

2+3

74+4-8

4+2

22+5

5+3

85+7

6+4

7+4

5+3

33+3

79+5

17+4

13+2

76+5

2+2

8+2

74 + 4

Mean ", + s.e.m.; determined by morphology and immunoperoxidase staining. BAL, bronchoalveolar lavage. Group A, HIV positive with Pneunmocvstiuc carinii pneumoniae (PCP); group Bl, HIV positive, PCP negative, with other infections; group B2, HIV positive, PCP negative, no infections; group C, HIV negative controls, no infections. *

DR expression The numbers of mature resident macrophages expressing DR were the same in all groups (Table 1). In the BAL samples of PCP patients, 85%, of cells with monocyte morphology expressed DR, whereas only 30% to 40% of peripheral blood monocytes expressed DR (data not shown). This increase in DR expression may be the result of maturation of these cells as they settle in the tissues or it may be induced by locally released cytokines.

500 r-

400 ~

I&

The effect of remotal of T cells In six experiments, macrophages from the samples were enriched on Nycodenz gradient. These were cultured in the wells in the same cell density as macrophages in unseparated samples (determined by differential counts using May-Grunwald/ Giemsa staining) and tested for TNF production. There was no

I T

300 E 0

TNF production by, BAL macrophages Stimulation of alveolar macrophages with LPS resulted in TNF-x release which was maximal at 24 h and started to decline by 48 h. Possible explanations for the decline are that it is either degraded or receptor-bound and internalized by macrophages. In subsequent experiments supernatants were collected at 24 h after initiation of cultures. The percentage of macrophages in the BAL samples varied significantly between the control and AIDS group. While the BAL fluid from control patients had 85-95°/O macrophages, those from AIDS patients had 25-80% of these cells. The number of macrophages present in the cultures were determined from total and differential counts and the values of TNF were expressed as U/ml per 105 macrophages. For this part of the study, patients from only group B2 were included. The BAL cells from AIDS patients with PCP (group A), in unstimulated cultures, produced significantly increased amounts of TNF-a compared with those from patients in groups B2 and C (Fig. 1). There was no significant difference in the LPSinduced TNF-i production between the groups.

T 200

z

100

(-

III

0

Group A

Group B2

Group C

Fig. 1. Spontaneous (0) and LPS-induced (-) TNF-a production by BAL macrophages. Bars are s.e.m. Mann Whitney U-test between group A and group B2 or group C, significant at 01'. For group criteria see Materials and Methods. M(D, macrophages.

difference in the levels of spontaneous and LPS induced TNF-ca release per 105 macrophages between those cultured in the presence of T cells (unseparated samples) and those cultured in their absence (enriched macrophages), ruling out the possibility of a direct T cell effect. significant

The efJect of remot al of macrophages The BAL cells from patients with PCP had 20-60° T cells. Previous studies have shown that T cells are capable of producing TNF-a (Cuturi et al., 1987). To check the possibility that T cells in the unstimulated cultures were producing TNF, in four experiments these were separated from macrophages on nylon wool columns (Julius, Simpson & Herzenberg, 1973) and cultured separately in the presence and absence of concanavalin A (Con A) (20 pg/ml). TNF-a was not detected in the

159

Alveolar macrophages in AIDS

800r 600-I

0 2

0 -

E

T

cl

400

1D

T LL

Z

-

U-

z

2

200

0l I

Group A

0

1 Group B2

Group C

I

.

.

U m1 ;1 * 100 200 300 400 500 Pneurnocystis cysts/5 microscopic field

600

Fig. 2. Spontaneous (0) and LPS-induced (U) TNF-a production by peripheral blood monocytes. Bars are s.e.m. Mann-Whitney U-test between group A and group B2 or group c; significant at 1 %.

Fig. 3. Comparison between the levels of TNF produced spontaneously in the cultures of BAL cells from PCP patients and Pneumocystis load in the fluid.

unstimulated lymphocyte cultures but significant levels were detected in mitogen-stimulated cultures. This confirms that the T cells are not the source of TNF produced spontaneously in the unseparated, unstimulated cultures of BAL cells from AIDS patients, and that the macrophages are most likely to be responsible for this.

episode of PCP. This was not seen with the cells taken from AIDS patients with respiratory symptoms between episodes of PCP or from ARC patients. Similar findings were obtained with peripheral blood monocytes. TNF-a was not detected in the plasma of these patients; this may be due to the sensitivity of the assay or rapid clearance of TNF from the circulation. It is also possible that this activity is confined to the lungs in vivo since this is usually the only organ affected by Pneumocystis. It is not clear whether the source of TNF-a produced are the alveolar macrophages or the blood-derived monocytes which constituted 30% of the macrophage population in the BAL fluids of PCP patients. Immunofluorescence staining techniques to detect cytoplasmic TNF were insensitive and not successful in identifying the TNF-producing cells. What stimulates the alveolar macrophage in vivo is unclear, although there are several candidates in the AIDS setting. HIV itself has been reported to induce TNF-ax release from T cells in vitro (Vyakarnam et al., 1988) but its effect on macrophages has not been studied. In a separate study using polymerase chain reaction technique for the detection of HIV-1 in lung macrophages, we detected HIV-cDNA in the adherent lavage cell population in five out of 10 patients with PCP and in five out of nine patients without PCP (Clarke et al., submitted). Since the alveolar macrophages from only the patients with PCP produced increased spontaneous TNF, it is unlikely that this is due to a direct effect of HIV. Although bacterial endotoxin is the prime inducer of TNF release from macrophages, other agents such as influenza virus and Sendai virus have also been found to induce TNF release (Beutler et al., 1986b; Aderka et al., 1986). IFN-y, Cornebacterium parvum, Mycobacterium bovis BCG and Plasmodium vinckei are known to activate macrophages and to increase the sensitivity to endotoxin, thereby augmenting TNF release (Beutler et al., 1986a; Clarke, Allison & Cox, 1976). From our data, Pneumocystis itself seems the most likely direct or indirect stimulus for TNF-a release by macrophages, since no other bacteria or viruses were found in the BAL fluids of all but two patients with PCP. The high levels of endotoxin present in some samples may have been responsible for inducing TNF release but do not account for the overall findings. Preliminary experiments attempting to use Pneumocystis to provoke TNF release from macrophages in short-term cultures were unsuc-

TNF-a production by peripheral blood monocytes Peripheral blood monocytes from AIDS patients with PCP, in unstimulated cultures, produced significantly increased amounts of TNF-a compared with those from patients in groups B2 and C (Fig. 2). Again there was no significant difference in the LPS-induced TNF release. The results showed that monocytes from patients in all groups were generally less efficient than alveolar macrophages in producing TNF in response to LPS. TNF was not detected in the plasma of patients with PCP. Endotoxin levels in samples This assay was kindly done by Dr K. F. Bayston at the Royal Postgraduate Medical School, London. Of 24 BAL samples from PCP patients tested for endogenous endotoxin, 10 had less than 50 pg, seven had 50 pg to 1 ng of endotoxin/ml, and five samples had endotoxin levels above 1 ng/ml. Spontaneous TNF-c synthesis in some but not all of the samples may have been due to high levels of endotoxin. Interestingly, BAL samples with levels of spontaneously produced TNF higher than 200 U/ml contained very low numbers of Pneumocystis cysts (less than 50 cysts per 5 microscopic fields). In contrast, TNF levels lower than 200 U/ml were associated with large numbers of cysts (100 to 500 cysts per 5 microscopic fields) (Fig. 3). The timing of bronchoscopy or the period of treatment with anti-Pneumocystis drugs did not appear to have any correlation with the observed Pneumocystis load in the BAL samples of these patients. There was no correlation between the amounts of TNF-ac spontaneously produced in the cultures and the patients' lung function tests such as transfer factor and blood gas measurements.

DISCUSSION We have demonstrated a spontaneous TNF-ax production in vitro by alveolar macrophages in AIDS patients during an

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CAPUT, D., BEUTLER, B. & HARTOG, K. (1986) Identification of a common nucleoyide sequence in the 3' untranslated region of the

CLARKE, I.A., ALLISON, A.C. & Cox, F.E. (1976) Protection of mice against Babesia and Plasmodium with BCG. Nature, 259, 309. CLOUSE, K.A., POWELL, D., WASHINGTON, I., POLI, G., STREBEL, K., FARRAR, W., BARSTAD, P., KOVACS, J., FAUCI, A.S. & FOLKS, T.M. (1989) Monokine regulation of human immunodeficiency virus 1 expression in a chronically infected human T cell clone. J. Immunol. 142, 431. CUTURI, M.C., MURPHY, M., GIOMI, M.P.C., WEINMAN, R., PERUSSIA, B. & TRINCHERI, G. (1987) Independent regulation of TNF and Lymphotoxin production by human peritoneal T cells. J. exp. Med. 165, 1581. EALES, L.J., MOSHTAEL, 0. & PINCHING, A.J. (1987) Microbicidal activity of monocyte derived macrophages in AIDS and Related disorders Clin. exp. Immunol. 67, 227. JULIUS, M.H., SIMPSON, E. & HERZENBERG, L.A. (1973) A rapid method for the isolation of functional thymus derived murine lymphocytes. Eur. J. Immunol. 3, 645. MEAGER, A., PARTI, S., LEUNG, H., PEIL, E. & MAHON, B. (1987) Preparation and characterisation of Monoclonal antibodies directed against antigenic determinants of Recombinant human TNF. Hybridoma, 6, 305. MURRAY, H.W., GELLENE, R.A., LIBBY, D.M., ROTHERMEL, C.D. & RUBIN, B.Y. (1985) Activation of tissue macrophages from AIDS patients: in vitro response of AIDS alveolar macrophages to lymphokines and interferon-gamma. J. Immunol. 135, 2374. PHILIP, R. & EPSTEIN, L.B. (1986) Tumour necrosis factor as immunomodullator and mediator of monocyte cytotoxicity indiced by itself, gamma-interferon and interleukin- 1. Nature, 323, 86. PINCHING, A.J., MCMANUS, T.J., JEFFRIES, D.J., MOSHTAEL, O., DONAGHY, M., PARKIN, J.M., MUNDAY, P.E. & HARRIS, J.R.W. (1983) Studies of cellular immunity in male homosexuals in London. Lancet, ii, 126. PRINCE, H., MOODY, D., SHUBIN, B. & FAHEY, J.L. (1985) Defective monocyte function in acquired immunodeficiency syndrome: evidence from a monocyte dependent T cell proliferative system. J. clin. Immunol. 5, 21. ROUX-LOMBARD, P., MODOUX, C., CRUCHAUD, A. & DAYRE, J.M. (1987) Increased tumour necrosis factor production by monocytes from patients with AIDS and ARC. Immunobiology, 175, 144. SCHEURICH, P., THOMA, B., UCER, U.C. & PFIZENMAIER, K. (1987) Early events during primary activation of T cells: antigen receptor crosslinking and interleukin 1 initiate proliferative response of human T cells. Eur. J. Immunol. 15, 1091. SILBERSTEIN, D.S. & DAVID, J.R. (1986) Tumour necrosis factor enhances eosinophil toxicity to Schistosoma mansoni larvae. Proc. nail Acad. Sci. USA, 83, 1055. SMITH, P.S., OHURA, K., MASUR, H., LANE, H.C., FAUCI, A.S. & WAHL, S.M. (1984) Monocyte function in acquired immune deficiency syndrome: defective chemotaxis. J. clin. Invest. 74, 2121. TAVERNE, J., DOCKRELL, M. & PLAYFAIR, J. (1981) Endotoxin induced serum factor kills malarial parasites in vitro. Infect. Immun. 33, 83. VILCEK, J., PALOMBELLA, V.J. & DESTEFANO, D.H. (1986) Fibroblast growth enhancing activity of TNF and its relationship to other polypeptide growth factors. J. exp. Med. 163, 632. VYAKARNAM, A., McKEATING, J., MEAGER, T. & BEVERLEY, P.C.L. (1990) Tumour necrosis factor induced by human immunodeficiency virus-i (HIV-I) in CD4 lymphocytes potentiates virus replication.

m-RNA molecules specifying inflammatory mediators. Proc. nail Acad. Sci. USA, 83, 1670. CARSWELL, E., OLD, L.J., KASSEL, R.C., GREEN, S., FIORE, N. & WILLIAMSON, B. (1975) An endotoxin induced serum factor that causes necrosis of tumours. Proc. Nail Acad. Sci. USA, 72, 3666.

AIDS, (In press). WALLACE, J.M., BARBERS, R.G., OISHI, J.S. & PRINCE, H. (1984) Cellular and T- lymphocyte subpopulation profiles in bronchoalveolar lavage fluid from patients with acquired immunodeficiency syndrome and pneumonitis. Am. Rev. resp. Dis. 130, 786.

cessful but further studies are required to substantiate a direct effect. It is possible that Pneumocystis has a priming effect on the macrophages so that they become more sensitive to low levels of endotoxin. Alternatively, Pneumocystis may affect other cells such as lymphocytes, and this might lead to a substantial change in the alveolar macrophage functional response. If the Pneumocystis quantification in the BAL fluids used in this study was a true reflection of the absolute cyst load in the lungs, then the association of higher TNF levels with low cyst counts could suggest a role for macrophage derived TNF-ot in the control and elimination of Pneumocystis carinji infection. TNF-a is already known to have indirect anti-parasitic effects: the killing of larval stage of Schistosoma mansoni by eosinophils is known to be enhanced by recombinant TNF (Silberstein & David, 1986). Endotoxin-induced serum factor killed malarial parasites in vitro (Taverne, Dockrell & Playfair, 1981). Autocrine activation of macrophages by locally produced TNF may have an important role in the direct or macrophage mediated killing of Pneumocystis. The recent evidence indicating that TNF-a may enhance HIV replication in infected lymphocytes (Clouse et al., 1989) suggests that TNF-cx release during episodes ofPCP may influence the natural history ofAIDS itself. Further studies may help to elucidate the role of TNF in the pathogenesis and control of Pneumocystis carinii infection.

ACKNOWLEDGMENT This work was supported by the British Lung Foundation.

REFERENCES ADERKA, D., HOLTMAN, H., TOKER, L., HAHN, T. & WALLACH, D. (1986) Tumour necrosis factor induction by Sendai virus. J. Immunol. 136, 2938. AMMAN, A., PALLADINO, M.A., VOLBERDING, P., ABRAMS, D., MARTIN, M.L. & CONANT, M. (1987) TNF-alpha and beta in AIDS and AIDS related complex. J. clin Immunol. 7, 6. BACHWICH, P., CHENSUE, S., LARRICK, J. & KUNKEL, S. (1986) Tumour necrosis factor stimulates interleukin-1 and prostaglanding E2 production in resting macrophages. Biochem. Biophys. Res. Commun. 136, 94. BANCROFT, J.D. (1975) Manual of Histological Techniques. (ed. by C. C. Harry & J. D. Bancroft) p. 212. Churchill-Livingstone, London. BEUTLER, B., KACENTO, U.T., MILSARK, I., KROCHIN, N. & CERAMI, A. (1986a) The effect of Interferon-Gamma on cachectin expression by mononuclear phagocytes. J. exp. Med. 164, 1791. BEUTLER, B., KROCHIN, N. & MILSARK, W. (1 986b) Induction of tumour necrosis factor synthesis by influenza virus. Clin. Res. 34, 491. BEUTLER, B., MAHONEY, J., LE TRANG, N., PEKALA, P. & CERAMI, A. (1985) Purification of cachectin, a lipoprotein lipase suppressing hormone secreted by endotoxin-induced RAW 264.7 cells. J. exp. Med. 161. 984.