Rosette formation by insect macrophages inhibition by cytochalasin B

CELLULAR

IMMUNOLOGY

29, 331-336

(1977)

Rosette Formation by Insect Macrophages Inhibition by Cytochalasin B ~IQBERT

Sloan-Kettrriug

Znstiflrtc

S. ANDERSON

for Carlcer Resrarclz, Donald S. IVuikrr

145 llostolt

Laboratory,

Post Road, Rye, hTrw I’orf~ IO.%‘0 Reccivcd

October 13,1976

Macrophages from the insect Spodopfcra cridania possess membrane receptors for unmodified avian and mammalian erythrocytes, with which they form spontaneous rosettes. Rosette formation occurs in the absence of serum proteins and divalent cations. Individual

macrophages

bear receptors

for several types of red cells. The level

of naturally-occurring hemagglutinins against a particular test erythrocyte is not correlated with macrophage reactivity against that red cell. In contrast with mammalian macrophages, neuraminidase treatment of either hemocytes or erythrocytes does not cause a marked enhancement of binding. Pretreatment of macrophages or erythrocytes with cytochalasin B causes reversible inhibition of rosetting probably by interfering with normal microfilament function, suggesting that optimal binding occurs when membranes are functioning normally on both macrophages and red cells.

Colchicine and vinblastine probably

not involved

do not influence rosetting ; therefore, microtubules are

in erythrocyte

binding.

ISTRODUCTION The phenomenon of the spontaneous production of nonimmune rosettes by mammalian T-lymphocytes and macrophages after incubation with erythrocytes has been studied extensively. In this study, the ability of insect (Spodofitera evidania) macrophages to form rosettes with various mammalian and avian erythrocytes is assayed.

Sjodoptera

hemolymph contains at least three different types of hemocytes: a

small cell having a prominent nucleus and scanty cytoplasm, and two larger cell types, one contains granules and the other is of hyaline character. The small cells

are progenitor cells (prohematocytes) which resemble lymphocytes, but this is merely a morphological similarity. The large cells are generally ovoid when in suspension, but readily attach to foreign surfaces upon which they spread, assuming varied configurations. The granular hemocytes appear to be involved in coagulation reactions and are not rosette forming. The hyaline hemocytes are highly phagocytic, comprise the rosetting cells, and are the cells referred to as macrophages in this report. Functionally and metabolically these phagocytes are more similar to mammalian macrophages than they are to polymorphonuclear leukocytes (1, 2). Insect macrophages have been shown to avidly phagocytize and/or encapsulate bacteria, erythrocytes and diverse foreign particulates (3). They are thought to play a significant role in controlling bacterial infections and in primitive celhllar immune reactions. 331

Copyright@ 1977by AcademicPress,Inc. All rights of reproductionin any form reserved.

ISSN 0008-8749

332

ROBERT

S.

ANDERSON

Cytochalasin B has been shown to inhibit reversibly rosette formation by human T-lymphocytes, presumably by disruption of microfilaments (4, 5). However, colchicine and vinblastine, which inhibit microtubular function, were shown to have little effect on human T-lymphocyte rosetting. In this paper the effects of those substances on the rosetting capacity of insect macrophages will be reported. Bentwich et al. (6) showed that pretreatment of lymphocytes with neuraminidase causes marked enhancement of erythrocyte binding. Kersey et al. (4) also reported this effect and that neuraminidase treatment abolished the temperature dependence of red-cell rosetting. In addition, the binding of neuraminidase-treated human erythrocytes is thought to be a specific property of human T-cells (7). Here is discussed the effect of neuraminidase treatment of insect macrophages on their rosetting capacity and their ability to bind neuraminidase-treated red cells. MATERIALS

AK11 METHODS

Terminal instar larvae of the Southern armyworm Spodoptcra cridanin we!-e used for all experiments. Hemocytes were obtained by bleeding via a proleg. Hemolymph was collected and maintained at about 1°C (on ice) and was immediately diluted with an equal volume of Ca?+ and Mg’+-free Dulbecco’s phosphatebuffered saline (PBS) pH 7.4 containing 05% ethylenediamine tetraacetic acid (EDTA). Rosette Formation

Rosettes were formed during 60 min incubation at 1°C of 0.1 ml of hemocyte suspension (5 x 10” cells) in PBS with 0.1 ml of 0.57 0 mammalian or avian erythrocytes. Mammalian and avian erythrocytes were obtained from the Animal Blood Centre, Inc. (Syracuse, New York). The incubation mixture was gently mixed after GO min, and the hemocytes counted in a haemocytometer; macrophages were counted as positive if three or more red cells adhered to their membranes. Cytochalasin B (Aldrich Chemical Company, Milwaukee, 1Visconsin) was maintained as a stock solution (10 mg/ml dimethylsulphoxide) and diluted in PBS prior to use. Colchicine and vinblastine (Sigma Chemical Company, St. Louis, Missouri) were dissolved directly in PBS. Solutions containing various concentrations of these drugs were added to the hemocyte suspensions, incubated GO min at l”C, and the hemocytes tested for rosetting capacity, as outlined above. To determine the reversibility of the cytochalasin B effect, the hemocytes were incubated for 60 min in 50 pgCB/ml PBS, washed twice and resuspended in PBS. The pretreated cells were then reacted with erythrocytes at various times after washing. In all cytochalasin B experiments, cell viability was tested by trypan blue exclusion. Ncwaminidme

Treatment

Fresh sheep erythrocytes were washed three times in saline and adjusted to a 0.5% suspension in PBS. The red cells were incubated with test concentrations of Vibrio cholerae neuraminidase (Behring Diagnostics, Somerville, New Jersey) for 60 min at 37°C. After incubation, the cells were washed three times and resuspended in PBS containing 0.1 s bovine serum albumin, at the final concentration of 1 x lO~/ml.

ROSETTE

FORMATION

RY

INSECT

TABLE Eiiect

of Cytochalasin Formation

Mean y0 rosettes

Rosette

RBC

Sheep RBC

Goose RBC

31.8 rt 4.3

27.0 zk 4.3

39.0 + 1.2

30.8 23.0 21.6 16.2 12.2

24.7 16.7 10.3 6.3 5.6

36.5 28.6 23.0 23.0 21.5

Human

None Cytochalasin 0.1 1.0 10.0 50.0 100.0

1

B on Mammalian and Avian Erythrocyte by Spodoptera eridania Macrophages

Treatment

333

M.4CROPIIAGES

B (@g/ml)

=t standard

deviation

zt zk f A f.

8.1 1.6 3.2 1.9 2.4

f * f f +

2.1 2.2 1.7 0.5 0.7

f f f f + -.

1.5 0.6 0.3 0.8 0.5 ~

..-

(II = 6).

Hemocytes were incubated with various concentrations of neuraminidase for 15-60 min at l”C, centrifuged and resuspended in PBS. The viability of all cell preparations was determined by trypan blue exclusion. These treated cells were then reacted with sheep and human red cells to produce spontaneous rosettes. RESULTS Spodoptrva macrophages were found to form rosettes with erythrocytes (E) from all mammalian and avian species tested, including guinea pig, rabbit, horse, rat, sheep, human, turkey and goose. The percent of cells forming rosettes with these E ranged from about 25-40%; Rosetting was carried out at 1“C because considerable hemocyte coagulation regularly occurred at higher temperatures. SI,odojtwa hemolymph contains weak naturally-occurring hemagglutinin (s) against rat and rabbit E, but this effect was not seen in these preparations because of the high final dilution of the hemolymph. As mentioned above, the h\-aline hemocytes (macrophages) are the only cells that produce E rosettes in these studies. There is no evidence for the existence of a nonuniform distribution of E receptors among subpopulations of these hemocytes. Mixtures of avian and mammalian E, which are easily morphologically differentiated from each other, were incubated with Spodoptcm macrophages. Spontaneous rosettes composed of sheep and goose E, and human and goose E, were readily produced. This suggested that individual insect macrophages bear receptors for more than a single type of red cell. A concentration-dependent reduction of human, sheep and goose E (HRBC, SRBC, GRBC) binding cells was observed after incubation with cytochalasin B (Table 1). Exposure to cytochalasin B ( < 100 pg/ml) did not effect cell viability, as indicated by trypan blue exclusion. Dimethylsulphoxide, at the concentration present in medium containing 100 pg/ml cytochalasin 13, had no effect on cell viability and did not cause any inhibition of rosette formation. Cytochalasin B (CB) inhibition of rosetting was shown to be reversible (Table 2) ; macrophages preincubated with 50 pg/ml CB were shown to regain gradually their rosetting capacity. The effects of preincubation of E with CB were also studied. Human E were incubated 60 min at 37°C with 100 pg/ m 1, washed and resuspended in PBS prior to reacting them with Spodoptrrn hemocytes. The CB-treated E showed markedly reduced rosetting reactivity (IS.5 + 36% of control values).

334

ROBERT

S. ANDERSON

Incubation of the macrophages with vinblastine at concentrations of lo-‘-lo-” &I, or colchicine at the same range of concentrations, had no significant effect on the number of HRBC or SRBC rosette-forming cells. These drugs at the higher concentrations cause diminution of human T lymphocyte viability. This effect was not seen in insect hemocytes; > 957’o viability, by trypan blue exclusion, was observed after exposure to either of the drugs at 10.” M. In no instance was the degree of binding of neuraminidase-treated SRBC to Spodoptera hemocytes significantly different from that of untreated erythrocytes. In these experiments the red cells were incubated with 50-150 U Y&io clzol~rae neuraminidase, washed and reacted with the macrophages. Hemocytes were incubated with lo-150 U neuraminidase, washed and tested for altered rosetting capacity. The results of this treatment were variable. One hour afteY washing, treated samples showed little change in the number of rosette-forming cells. Two hours after treatment, the experimental preparations generally contained more rosetting cells than, the controls; however, the effect was not dose-dependent. DISCUSSION This paper describes the formation of spontaneous, nonimmune mammalian and avian erythrocyte rosettes by a population of insect leukocytes analogous to macrophages. In mammals, thymocytes and thymus-derived cells (T cells) can bind to autologous and heterologous red cells ; this reaction occurs in mice (a), rabbits (9) and human beings (7, 10). Spontaneous macrophage-red cell rosette formation has been observed in many mammalian species (11). Rlacrophages were shown to have receptors for unmodified red cells of various other species. Rosetting by macrophages was shown not to require Ca*+ or lVlg2+ since final EDTA concentrations as high as 1% did not inhibit the reaction, A low concentration of EDTA (0.576) was present in the medium to inhibit hemocyte clumping ; however, rosetting proceeded 6ell under these conditions. Lalezari et al. (11) reported that rosetting by mammalian macrophages occurred with fully washed cells in the absence of exogenous serum proteins and that there was no correlation between rosetting and the level of preexisting circulating heterophilic anti-red cell antibodies. The results with insect macrophages are similar in that resetting occurred around thoroughly washed cells in the absence of humoral factors in the medium. Little naturally-occurring hemagglutinating activity against the red cells used in the rosetting studies was detected in Spodofitera hemolymph. Other studies have shown that, while insects possess natural hemagglutinins, these factors have little or no opsonic activity (12, 13). TABLE Keversibility

of Cytochalasin RBC

B Inhibition Recovery

of Rosetting

by Spodopteru

(70 control rosetting

0 min* Sheep Human Goose

2

19.1 f 4.4 45.7 f 3.8 60.8 f 2.2

& standard

60 min 87.9 f 85.2 f 84.0 f

* Time after hemocytes were washed free of cytochalasin resuspended in cytochalasin B-free medium.

0.8 2.3 0.8

erddaniu

Macrophages

deviation,

n = 5)

120 min 100.0 93.6 f 93.2 f

3.2 0.7

B (50 ,ug/ml, 60 min incubation)

and

ROSETTE

FORMATION

BY

INSECT

MACROPFIAGES

335

Neuraminidase treatment of T lymphocytes enhanced erpthrocyte receptor activity (4, 6). The marked increase in rosetting caused by such preincubation could not be shown after incubation of Spodoptcra macrophages with Vibrio ckolerae neuraminidase (VCK) . Reaction of red blood cells with neuraminidase will promote their binding to human T cells (7). However, VCN-treated erythrocytes are bound to about the same extent as untreated E by insect hemocytes. In recent years, attention has been drawn to the apparent association between cell surface movement (and binding reactions) and 70 A diameter contractile microfilaments. A network of these microfilaments is present in the peripheral cytoplasm of macrophages (14). Many of the microfilaments are actin-like and bind heavy meromyosin, and some may be inserted into the plasma membrane. Microfilaments are thought to be involved in cell movement, maintenance of normal cell morphology, r&fled membrane movement, phagocytosis, receptor mobility, and the binding of erythrocytes to leukocytes during rosette formation. The role of microfilaments in cellular and developmental processes and the inhibition of their function by cytochalasin B was reviewed by Ij’essels et 01. (15). The effects of cytochalasin B on eukaryotic cells are numerous and involve inhibition of sugar transport (16, 17)) as well as microfilament clepolymerization. The effect of CB on glucose transport occurs at much lower concentrations than those required for microfilament disruption. In the case of human red blood cells, there are both high and low affinity binding sites for CB. Current evidence indicates that the high affinity receptors are related to sugar transport (IS), while the low affinity sites affect microfilaments. The mold metabolite cytochalasin B, at concentrations which interfere with microfilament function in mammalian cells (16)) reversibly inhibits binding of erythrocytes by macrophages of the insect Spodopfeva. Cytochalasin B has a similar inhibitory effect on the formation of human T-lymphocyte red cell rosettes (4, 5)) presumably by depolymerization of microfilaments. Human T cells develop short microvilli during SKBC rosetting reactions (1921). Inhibition of microfilaments by cytochaIasin B would in turn inhibit microvilli formation which is probably required for leukocyte-erythrocyte binding. The effect of cytochalasin B on the ultrastructure of mouse peritoneal macrophages has been studied (22). A disorganization of cortical microfilaments and increased zeiosis (blebbing) was induced in these macrophages by cytochalasin B. Vinblastine and colchicine interact with microtubules causing their disintegration (23, 24). These drugs do not inhibit human T-lymphocyte rosette formation (4, 5). Therefore, it would appear that these cytoskeletal elements do not play a role in this phenomenon. These data suggest the same conclusion in the case of erythrocytes rosetting by Spodoptcra macrophages, since neither vinblastine nor colchicine interferes with the process. Furthermore, microtubules are temperaturesensitive, disappearing at low temperatures (23). Insect macrophages from red cell rosettes at 1“C, further supporting the idea that microtubular function is not required for E binding. Red blood cell rosetting by mammalian T lymphocytes is also observed at low temperatures (7, 25, 26). These studies show that erythrocyte receptors on leukocytes are found in invertebrate animals such as insects, as well as in mammals. The phenomenon of red cell rosetting by vertebrate macrophages and T lymphocytes has bee11 extensively studied. Spodoptera macrophages (insects lack lymphocytes) are ca-

336

ROBERT

S. ANDERSON

pable of forming typical rosettes with avian and mammalian erythrocytes. Conditions for binding of red cells to blood cells from these phylogenetically disparate animals showed a number of similarities including no requirements for divalent cations, exogenous serum proteins, or cell-bound humoral recognition factors. Other vertebrate macrophage reactions require cytophilic antibodies and/or complement ; these reactions have not been demonstrated in the case of insect macrophages. Red cell rosetting by lymphocytes has been shown to be dependent on normal microfilament functioning, on the basis of cytochalasin B studies. A similar, dose-dependent, reversible inhibition of rosetting by Spodoptcra macrophages treated with cytochalasin B is reported here. The microtubule-disrupting drugs, vinblastine and colchicine, have no effect on red cell rosetting by both mammalian and insect leukocytes. ACK?;OM’LEDGMENTS The Boyce Thompson Institute, Yonkers, New York, generously contributed the animals used in this study. Miss Molly Cook provided excellent technical assistance. This research was supported by a grant from the Whitehall Foundation and CA-08748 from the National Cancer Institute.

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