Influence of Autologous Monocytes on the Pokeweed Mitogen-Induced Generation of Immunoglobulin-Secreting Cells in Man

Scand J Haematol(l982) 29,273-282

Influence of Autologous Monocytes on the Pokeweed Mitogen-Induced Generation of Immunoglobulin-Secreting

Cells in Man

J 0 R G E N PETERSEN, MARIANNE KIEFFER, DESA LILIC, NIELS RATHLEV & VAGN ANDERSEN

Laboratory of Medical Immunology, Department of Medicine TA, Rigshospitalet, Copenhagen, Denmark

The regulatory influence of autologous blood monocytes on the PWM-induced generation of Ig-secreting cells was assessed using a reverse haemolytic plaque forming cell (PFC) assay. The PWM-induced PFC responses of monocyte-depleted cells were low or absent in most cases. Addition of 1242 % freshly isolated monocytes fully reconstituted the IgM-, IgG- and IgA-PFC responses. With more monocytes added, the PFC responses declined. Monocytes precultured for 48 h supported the PFC responses of monocyte-depleted cells less well and addition of monocytes stimulated with phorbol myristate acetate (PMA) did not increase the response. The low responses of monocyte-depleted cells co-cultured with precultured monocytes were not increased by addition of supernatant from monocyte cultures. The PFC responses of mononuclear cells induced by PWM were significantly inhibited by unstimulated precultured monocytes, and to a larger degree by PMA-treated monocytes, indicating the presence of suppressor cells among the precultured monocytes. The PWM- induced thymidine incorporation by monocyte-depleted cells with precultured monocytes added was only slightly lower than that obtained with freshly isolated monocytes added, suggesting that the suppressive role of precultured monocytes was not due to cytotoxicity.

Key words: B lymphocyte activation - immunoglobulin-secreting cells - monocytes/macrophages

Accepted for publication March 1,1982

Correspondence to: Dr. Jprgen Petersen, Laboratory of Medical Immunology TA 7544, Rigshospitalet, Tagensvej 20, DK-2200 Copenhagen N, Denmark

ABBREVIATIONS FCS = fetal calf serum LPS = lipopolysaccharide MEM = minimum essential medium PFC = plaque forming cell PMA = phorbol myristate acetate PWM = pokeweed mitogen SRBC = sheep red blood cells SPA = S. aureus protein A

During recent years, the establishment of reverse haemolytic plaque forming cell assays for visualizing Ig-secreting cells has lead to increased understanding of the factors regulating B lymphocyte activity in man. Monocytes are important for the responses of lymphocytes. The presence of these cells has been shown to be necessary

0036553X/82/090273-10 $02.50/0 Q 1982 Munksgaard, Copenhagen

274 PETERSEN, KIEFFER, LILIC, RATHLEV & ANDERSEN

for proliferation and differentiation of T lymphocytes (Rosenstreich et a1 1976, Op- penheim & Rosenstreich 1977) and of B lymphocytes (Blaese et a1 1977).

In a number of disease conditions evidence for suppressive effects of monocytes on various lymphocyte functions has been provided. Monocytes with suppressor activity have been demonstrated in patients with malignancies, including Hodgkin's disease (Goodwin et a1 1977) and multiple myeloma (Broder et a1 1975), and in patients with systemic lupus erythematosus (Markenson et a1 1978) and sarcoidosis (Katz & Fauci 1978).

Monocytes cultured in vitro differentiate into mature macrophages (0degaard et a1 1974). A number of reagents have been employed to enhance the 'activation' of cultured macrophages, including bacterial lipopolysaccharide (LPS), antigen-antibody complexes and phorbol myristate acetate (PMA) (Gery & Waksman 1972, Unanue et a1 1976, Blyden & Handschumacher 1977, Mizel et a1 1978). The purpose of the present study is to investigate the regulatory role of monocytes for the generation of Ig-secreting cells as induced by pokeweed mitogen (PWM). The regulatory properties of monocytes are compared to those found using monocytes precultured in vitro.

MATERIAL AND METHODS

Isolation of mononuclear cells Heparinized blood from healthy adult volunteers was separated on Lymphoprepm (Nyegaard, Oslo, Norway). The mononuclear cells were washed and resuspended in RPMI 1640 (Gibco, Paisley, Scotland) supplemented with 10% fetal calf serum (FCS) (Flow, Irvine, Scot- land) and L-glutamine (0.8 mmol/l) plus penicillin (500 IU/ml), streptomycin (500 pg/ml), and nystatin (20 U/ml).

Preparation of monocyte-depleted and monocyte- enriched suspensions Mononuclear cells (4 x 106/ml) were incubated in Petri dishes (diameter 60 mm, Falcon No. 3002, Oxnard, Calif., USA) in a vol of 1.5 ml for 90 min at 3 P C . Non-adherent cells were decanted, and the dishes were washed 5 times with 37°C RPMI with 1% FCS. Ad- herent cells were detached by incubation with EDTA (10 mmol/l in PBS) at 4°C for 20 min, and by scraping with a rubber policeman, and then washed and kept at 4°C until use. Monocytes were quantified by staining cytocentrifuged smears for a-naphthyl acetate esterase activity (Horwitz et a1 1977).

Monocyte cultures Adherent cells were cultured in the Petri dishes in RPMI 1640 with 10 % FCS. In some experiments, phorbol myristate acetate (PMA) (Sigma, St. Louis, Miss., USA) was added at the onset of culture in a concentration of 0.8 x 1k6 mol/l. Ph4A was dissolved in 99 % ethanol; the final concentration of ethanol in the cultures did not exceed 0.1 g/l. After 48 h at 37°C the dishes were washed 5 times and adherent cells were detached as above. Cell-free monocyte supernatant was stored at -20" C.

Induction of the PFC response to PWM Mononuclear cells were cultured at 1.0 x 106 cells/ml in flat-bottomed microtiter plates (Nunc, Roskilde, Denmark), 0.25 ml per chamber. Mixtures of cells were always performed on the basis of viable cells; these were quantified by nigrosin exclusion. At least triplicate experiments were carried out. Pokeweed mitogen (PWM) (Gibco) was added at a concentration of 0.8 pg/ml. Cells were incubated for 6 d at 37°C in a humidified atmosphere of 95% air and 5% C q . At harvest, cells were washed once in minimum essential medium (MEM) (Flow) prior to plating.

PFC assay The PFC assay used is a modification of the method described by Hammarstrom et a1 (1979) and the assay is described in detail elsewhere by Agger et a1 (1982). In brief, S. aureus protein A (SPA) (Pharmacia, Upp- sala, Sweden) (0.5 mg/ml in 0.9% NaCI) was mixed with an equal volume of intensively washed sheep red blood cells (SRBC) and with 10 vol of CrCb (1 % w/v in 0.9 % NaCl, diluted 1:150) (Gronowicz et a1 1976). After incubation for 1 h at 3WC, SPA-conjugated SRBC were washed once in 0.9% NaCl and twice in MEM.

MONOCYTE REGULATION OF Ig-SECRETION 275

All reagents were diluted in MEM. 100 p1 mononuclear cells in a dilution appropriate for plaque counting, 50 pl SPA-SRBC diluted 1:6, 25 p1 rabbit anti-human IgM, IgG or IgA (DAKO, Copenhagen, Denmark) diluted 1:30, 25 p1 guinea pig complement (Flow) absorbed twice with SRBC and diluted 1:4 were mixed with 800 pl 0.5% Noble agar (Difco, Detroit, Mich., USA) supplemented with DEAE- dextran (0.45 mglml) (Pharmacia) at 45°C. 3 drops of 200 pl were placed without delay on the lid and the bottom of 9 cm Petri dishes (Nunc) and covered with 50 x 25 mm cover slips. The preparations were incubated for 4 h at 37°C and plaques were read at 10 x magnification using indirect light. The number of PFC was calculated per lo6 originally cultured cells.

Proliferative capacity In order to elucidate the extent of DNA synthesis in the cultures, cells were taken from the cultures on day 6 and incubated for 6 h at 37°C in a vol of 100 p1 with 0.05 pCi [14C]-thymidine (Sp.act. > 50 mCi/mmol). The cells were collected on glass fibre filters in a multiple cell harvester, and thymidine incorporation was estimated by liquid scintillation counting. The results given are medians of cpm measured in triplicate determinations, calculated per 105 originally cultured cells.

Statistics Results were analysed by the Mann-Whitney rank sum test. P values < 0.05 were regarded as significant.

RESULTS

Cellular composition In 15 experiments, freshly isolated mononuclear cells contained 16 f 6 % monocytes (mean f 1 s.d.) whereas monocyte-depleted cells contained 1.4 f 1.0% monocytes. The proportion of monocytes in monocyte-enriched suspensions was 72 f 10 %. In precultured monocyte-enriched suspensions and in PMA- treated monocyte-enriched suspensions the content of monocytes was 80 f 8% and 81 f 7 % respectively.

The viability of freshly isolated monocyte- depleted cells and of monocyte-enriched cells always exceeded 95 % whereas the viability of precultured unstimulated monocyte-enriched cells was slightly lower but always exceeding 85 %. PMA-treated monocyte-enriched suspensions contained approximately 60 % viable cells.

As compared to freshly isolated monocytes, the precultured monocytes (untreated or PMA-treated) showed morphological signs of differentiation into large macrophages as described by 0degaard et a1 (1974).

PWM-induced responses of mononuclear cells and monocyte-depleted cells The PFC responses to PWM by mononuclear cells and by monocyte-depleted cells are shown in Figure 1. PWM added to mononuclear cells elicited a vigorous PFC response within all 3 Ig-classes. By comparison, less than 1000 IgM-, IgG- and IgA- PFC/106 were generated by unstimulated cells.

As compared to unseparated cells, the PWM responses of monocyte-depleted cells were low in 8 of 9 experiments (Figure 1) (P < 0.05). In 1 of 9 experiments, however, a considerable response (approximately 50 % of that of unseparated cells) by the monocyte-depleted cells was noted (Figure 1). No correlation between the residual monocyte content of monocyte-depleted cells and the PFC responses obtained was observed.

Influence of freshly isolated monocytes on the PFC response induced by PWM As seen in Figure 2 ~ , addition of freshly isolated autologous monocytes increased the IgM-PFC response of monocyte-depleted cells dramatically. The optimal concentration of monocytes varied from 1242% in

276

(D 0 LOOOO- K 0 L L e

20000 -

1 60 000

.

. . . - - . . @ * . - .. 9 . . . e . . 0 * - .

I I I 1 1 I I I e .... 0 0 . . ..* w

PETERSEN, KIEFFER, LILIC, RATHLEV & ANDERSEN

A 0

0 .

IgM IgG I g A IgM IgG I g A -PFC -PFC -PFC -PFC -PFC -PFC

Figure 1, PWM-induced responses of unseparated mononuclear cells (A) and monocyte-depleted celIs (B) cultured for 6 d. PFC responses by monocyte-depleted cells were low or absent in most cases. Bars represent mean PFC/106 originally cultured cells.

different experiments; the optimal concentrations were equal for the generation of IgM-, IgG- and IgA-PFC (data not shown). At optimal concentration, the freshly isolated monocytes fully reconstituted the PFC responses of the monocyte-depleted cells.

A decline of the PFC response of all 3 Ig-classes was noted with concentrations of monocytes exceeding the optimal concentration (data shown in part in Figure 2 ~ ) .

Influence of precultured monocytes on the P WM-generated PFC response The PFC response of monocyte-depleted

cells, to which precultured monocytes had been added in varying proportions, is displayed in Figure 2 ~ . In comparison to freshly isolated monocytes, the precultured, unstimulated monocytes supported the induction of IgM-PFC by PWM less well or did not improve the response at all. Similar results were obtained in the case of IgG- and IgA-PFC (data not shown).

Monocytes stimulated with PMA did not support the PFC response of monocyte- depleted cells (data not shown). As monocytes and macrophages have been shown to release a number of soluble products (for

MONOCYTE REGULATION O F Ig-SECRETION 277

B

0 10 20 30 LO 50 60 70 0 10 20 30 LO 50 60 70

% MONOCYTES Figure 2. Effect of addition of autologous monocytes to monocyte-depleted suspensions. Following the addition of monocytes, the cells were stimulated with PWM for 6 d. Ordinate: Number of IgM-PFC/106 originally cultured cells. Abscissa: % monocytes among the PWM-stimulated cells. A: Addition of freshly isolated monocytes. B: Addition of monocytes precultured for 48 h.

review, see Rocklin et a1 1980), experiments were performed to investigate the influence of monocyte supernatants on the PFC response. Initial experiments showed that addition of monocyte supernatants to monocyte-depleted cells did not increase the PFC response induced by PWM (Ta- ble 1). In agreement with the findings of Dimitriu et a1 (1978), addition of monocyte supernatant to monocyte-depleted cells reconstituted with freshly isolated rnonocytes increased the PFC responses although the differences did not reach statistical signi- ficance. From the data presented in Table 1, it is apparent that rnonocyte-depleted cells with addition of precultured monocytes still

displayed poor PFC responses to PWM after addition of monocyte supernatant.

To determine whether the low PFC response of monocyte-depleted cells with the addition of precultured monocytes was due to the presence of suppressor cells among the precultured adherent cells, experiments were carried out as summarized in Table 2. Freshly isolated monocytes added to mononuclear cells in concentrations of 20 or 40% had no significant influence on the PWM- induced PFC formation. By contrast, addition of similar proportions of precultured monocytes significantly suppressed the IgM- PFC response (Table 2). With addition of PWM-treated monocytes, even more pro-


TABLE 1 PWM-induced PFC responses of mononuclear cells:

effect of addition of supernatant from a 48 h unstimulated monocyte culture'

+ 20 % freshly +20% monocytes % supernatant isolated precultured from a 48 h

rnonocytes for 48 h monocyte culture

Unseparated cells no no 0 17 220 ? 10 544

Monocyte-depleted cells no no 0 378 ? 198 no no 10 826 f 4732 no no 20 748 ? 4572

Monocyte-depleted cells no 0 13 087 k 3 319 no 10 18 756 -C 4 8172 no 20 15 864 k 424@

Monocyte-depleted cells no yes . 0 1718 k 1517 no Yes 10 2 292 k 1 9542 no Yes 20 1 809 ? 1 3312

1 Data are expressed as IgM-PFC/106 originally cultured cells (mean k 1 s.d. of 5 experiments). 2 Not significantly different from experiments with no addition of monocyte supernatant.

TABLE 2 PWM-induced PFC responses of unseparated mononuclear cells:

effects of addition of freshly isolated monocytes or precultured monocytes'

Proportions of rnonocytes added

0% 20 % 40%

Freshly isolated monocytes 12 414 14 7742 11 3722 1 1 J J

1 1 J J

P < 0.01

8963

P < 0.01

Unstimulated, precultured monocytes 12 414 3 0883

P < 0.05 n.s.4

2773 PMA-treated, precultured monocytes 12 414 4783

Data are mean IgM-PFC/106 originally cultured cells of 5 experiments.

P < 0.05 when compared to mononuclear cells with no addition of monocytes. n.s. = not significant.

* Not significantly different from PFC obtained with mononuclear cells cultured without addition of monocytes.

nounced suppression of IgM-PFC formation Influence of freshly isolated and precultured was seen. IgG- and IgA-PFC generation by monocytes on the PWM-induced unseparated mononuclear cells was likewise proliferation suppressed by unstimulated and by PMA- As demonstrated in Table 3, the PWM- treated precultured monocytes (data not generated thymidine incorporation by un- shown). separated cells was moderately reduced

MONOCYTE REGULATION O F Ig-SECRETION 279

TABLE 3 Thymidine incorporation and cell recovery after 6 d culture of unseparated mononuclear cells

and monocyte-depleted cells'

cpm/l05 cells Recovery (%)

Unseparated cells (n = 9) 428 f 193 48 ? 28 Unstimulated PW M-stimulated 1448 f 392 86 ? 33

38 f 20 P WM-stimulated 947 ? 340 54 ? 36

Monocyte-depleted cells (n = 9) Unstimulated 217 k 331

The results given are means ? 1 s.d. of 9 experiments.

A B 2000 -

m

500 -

0- I I 1 I I I I I I I 1 I I

0 10 20 30 LO 50 60 70 0 10 20 30 LO 50 60 70 O/o MONOCYTES

Figure 3. Effect of adding autologous monocytes to monocyte-depleted cells on thymidine incorporation after 6 d of culture with PWM. Ordinate: cpm/105 originally cultured cells. Abscissa: % monocytes added. A and B as in Figure 2.

after removal of monocytes before initiation addition of monocytes, and maximal thymi- of culture, and a similar reduction in cell dine uptake was obtained with concentra- recovery was observed. The effect of adding tions of monocytes ranging from 12-28 %. freshly isolated monocytes to monocyte- The thymidine incorporation declined with depleted cells before initiation of culture is the addition of more monocytes (Figure 3 ~ ) . outlined in Figure 3 ~ ; in most experiments, In most experiments, slightly reduced thymi- thymidine incorporation increased with the dine incorporation was found with addition


of precultured monocytes as compared to addition of freshly isolated monocytes (Fig- ure 38). Similar results were obtained with the addition of PMA-stimulated monocytes (data not shown).

DISCUSSION

The present investigation has confirmed that the presence of monocytes is a require- ment for the generation of Ig-secreting cells by PWM. In 8 of 9 experiments, the PWM- induced PFC responses were low or absent after removal of monocytes. Analogous results have been obtained by other groups using PFC formation (Rosenberg & Lipsky 1979), Ig secretion (Gmelig-Meyling & Wald- mann 1981) or cells with cytoplasmic Ig (Knap & Baumgartner 1978) as parameters for the PWM-driven lymphocyte activation. The PWM-induced PFC responses were fully reconstituted in all cases by the addition of freshly isolated monocytes at an appropriate concentration to monocyte-depleted cells.

A suppressive effect of freshly isolated monocytes on the PWM-induced PFC response was observed when monocytes were added at concentrations exceeding the optimal. When 50 % monocytes were added to monocyte-depleted cells, the PFC responses were low or absent in agreement with previous findings (Knapp & Baumgartner 1978, Gmelig-Meyling & Waldmann 1981). Recently, a similar suppressive action of monocytes added in excess on the Ig secretion induced by S. aureus, nocardia water soluble mitogen or Epstein-Barr virus has been demonstrated by Gmelig-Meyling & Waldmann (1981).

It has recently been reported by Rosen- berg & Lipsky (1981) and by our group in a

preliminary communication (Petersen et a1 198l), that monocytes subjected to pre- culture for 48 h do not increase the PWM response when added to monocyte-depleted cells, This observation was confirmed and extended by the present study. A similar but more pronounced effect was observed using PMA-treated monocytes. However, thymidine incorporation was only slightly af- fected, suggesting that the low PWM response was not due to a cytotoxic effect of the precultured monocytes. Addition of monocyte supernatant to monocyte-depleted cells co-cultured with precultured monocytes did not restore the PWM-induced response. Thus the marked difference between freshly isolated and precultured monocytes could not be ascribed to a loss of soluble products of importance for the PWM-induced response.

During in vitro culture, monocytes/macrophages are activated as determined by al- tered morphology ((ddegaard et a1 1974), increased synthesis of enzymes (Karnovsky & Lazdins 1978) and increased metabolic activity (Karnovsky & Lazdins 1978), paral- leling the findings in macrophages from in- flammatory foci in vivo. PMA has been used to enhance the in vitro activation of macrophages (Nathan & Root 1977, Vassalli et a1 1977, Mizel et a1 1978). The experiments of this study clearly showed that suppressor cells were generated during the culture of monocytes. This was evidenced by a significant inhibition of the PWM-induced PFC formation following addition of PMA-stim- dated monocytes in concentrations of 20 or 40 %. A lesser, but still significant suppression was obtained by addition of unstim- dated, precultured monocytes. The experiments thus indicate that the regulation of polyclonal B cell activation in vitro by freshly isolated and by precultured monocytes is different. However, the biochemical

MONOCYTE REGULATION OF &-SECRETION 28 1

mechanisms of the suppressive activity of precultured monocytes remain to be clarified.

ACKNOWLEDGEMENTS

This work was supported by the National League against Rheumatism and the Danish Medical Research Council. Mrs. Vita Weibull and Mrs. Anne Ambjflrn- sen are gratefully acknowledged for their skilful tech- nical assistance.

REFERENCES

Agger R, Petersen J, Dinesen B, Wiik A & Andersen V (1982) Production and secretion of immunoglobulins by in vitro-activated human B lymphocytes. Allergy 37, 179-85.

Blaese R M, Lawrence E C & Poplack D G (1977) A critique of techniques of macrophage-monocyte de- pletion in studies of human peripheral blood mononuclear leukocytes. In D 0 Lucas (ed) Regulatory mechanisms in lymphocyte activation, 579. Academic Press, New York.

Blyden G & Handschumacher R F (1977) Purification and properties of human lymphocyte activating factor (LAF). J Immunolll8, 1631-38.

Broder S , Humphrey R, Durm, M, Blackman -M, Meade B, Goldman C, Strober W & Waldmann T (1975) Impaired synthesis of polyclonal (non-para- protein) immunoglobulins by circulating lymphocytes from patients with multiple myeloma. N Engl J Med 293,887-92.

Dimitriu A & Fauci A S (1978) Activation of human B lymphocytes. IX. Modulation of antibody production by products of activated macrophages. J Im- munoll20, 1818-23.

Gery I & Waksman B H (1972) Potentiation of the T-lymphocyte response to mitogens. 11. The cellular source of potentiating mediator(s). J Exp Med 136, 143-55.

Gmelig-Meyling F & Waldmann T A (1981) Human B cell activation in vitro: augmentation and suppression by monocytes of the immunoglobulin production induced by various B cell stimulants. J Immunol 126,529-37.

Goodwin J S, Messner R P, Bankhurst A D, Peake G T, Saiki J H & Williams R C (1977) Prostaglandin- producing suppressor cells in Hodgkin’s disease. J Engl J Med 297,963-68.

Gronowicz E, Coutinho A & Melchers F (1976) A plaque assay for all cells secreting Ig of a given type or class. Eur J Immunol6, 588-90.

Hammarstrom L, Bird A G, Britton S & Smith C I E (1979) Pokeweed mitogen induced differentiation of human B cells: evaluation by a protein A haemolytic plaque assay. Imrnunol38, 181-89.

Honvitz D A, Allison A C, Ward P & Kight N (1977) Identification of human mononuclear leucocyte populations by esterase staining. Clin Exp Immunol 30,289-98.

Karnovsky M L & Lazdins J K (1978) Biochemical criteria for activated macrophages. J Immunol 121,

Katz P & Fauci A S (1978) Inhibition of polyclonal B-cell activation by suppressor monocytes in patients with sarcoidosis. Clin Exp Immunol32,554-62.

Knapp W & Baumgartner G (1978) Monocyte-mediated suppression of human B lymphocyte differentiation in vitro. J Immunoll21, 1177-83.

Markenson J A, Morgan J W, Lockshin M D, Joachim C & Winfield B (1978) Responses of fractionated cells from patients with systemic lupus erythematosus and normals to plant mitogens: evidence for a suppressive population of monocytes. Proc SOC Exp Biol Med 158,5-9.

Mizel S B, Rosenstreich D L & Oppenheim J J (1978) Phorbol myristic acetate stimulates LAF production by the macrophage cell line, P388D. Cell Immunol 40,230-35.

Nathan C F & Root R K (1977) Hydrogen peroxide release from mouse peritoneal macrophages: depen- dence on sequential activation and triggering. J Exp Med 146,1648-62.

Oppenheim J J & Rosenstreich D L (1977) Signals regulating in vitro activation of lymphocytes. Prog Allergy 20,65-194.

Petersen J, Agger R, Kieffer M, Lilic D, Rathlev N & Andersen V (1981) Immunoregulatory mechanisms evaluated by quantitation of immunoglobulin-secreting cells in man. In C M Steinberg & I Lefkovits (eds) The immune system, vol2, p 84. Karger, Basel.

Rocklin R E, Bendtzen K & Greineder D (1980) Mediators of immunity: lymphokines and monokines. Adv Zmmunol29,55-136.

Rosenberg S A & Lipsky P E (1979) Monocyte depen- dence of pokeweed-mitogen induced differentiation

809-13.


of immunoglobulin-secreting cells from human peripheral blood mononuclear cells. J Immunol 122, 926-31.

Rosenberg S A & Lipsky P E (1981) The role of monocytes in pokeweed mitogen-stimulated human B cell activation: separate requirements for intact monocytes and a soluble monocyte factor. J Immunol 126, 134145.

Rosenstreich D L, Farrar J J & Dougherty S (1976) Absolute macrophage deficiency of T lymphocyte activation by mitogens. J Immunol ll6, 131-39.

Unanue E R, Beller D I, Calderon J, Kiely J M & Stadecker M J (1976) Regulation of immunity and inflammation by mediators from macrophages. Am J Pathol85, 465-78.

Vassalli J-D, Hamilton J & Reich E (1977) Macrophage plasminogen activator: induction by concanavalin A and phorbol myristate acetate. Cell 11, 695-705.

Odegaard A, Viken K & Lamvik J (1974) Structural and functional properties of blood monocytes cultured in vitro. Acta Pathol Microbiol Scand Sect B 82,223-34.

Documents

Influence of Autologous Monocytes on the Pokeweed Mitogen-Induced Generation of Immunoglobulin-Secreting Cells in Man