Apparent adoptive immune enhancement by bone marrow and bursa of fabricius cells from juvenile chickens

DEVELOPMENTAL AND COMPARATIVE I~fl~UNOLOGY, Vol. 7, pp. 313-323, 1983. 0145-305X/83/020313-II$03.00/0 Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.

APPARENT ADOPTIVE IMMUNE ENHANCEMENT BY BONE MARROW AND BURSA OF FABRICIUS CELLS FROM J U V ~ I L E CHICKENS

Frank Seto Zoology Department University of Oklahoma Norman, Oklahoma 73019

ABSTRACT

The occurrence of enhancer cells (EC) in the bone marrow and bursa of Fabricius of unprimed and mouse erythrocyte (MRBC) primed birds was investigated. EC are defined operationally as immunocompetent cells that are incapable of adoptive immunity in embryo hosts but able to enhance the immune responsiveness of newly-hatched chick recipients. Weak to moderate immune enhancement was observed in chick hosts grafted with bursal cells from unprimed or MRBC-primed donors whereas with bone marrow cell transfer the immune enhancement was weak with cells from unprimed donors but modest to strong with those from primed donors. Thus antigen priming of donors had little effect on the EC level of the bursa but appeared to increase that in the bone marrow. Moreover, the EC activity of bone marrow of primed donors was donorage dependent. Response profile studies revealed that the EC level of donor bone marrow was low the first 2-3 days after immunization and high by day six. The elevated EC level in the bone marrow of immunized donors is believed due to both recruitment of immigrant cells of extramedullary origin and clonal expansion of medullary immunocompetent cells.

INTRODUCTION Cell transfer studies of lymphocyte suspensions from various chicken lymphoid organs revealed that some lymphoid cells are capable of adoptive immunity in histocompatible embryo hosts whereas others failed (1,2). Spleen cells and peripheral blood leucocytes from antigen-primed birds showed high adoptive immune capacity as measured by serum hemagglutinin titers (3) and splenic PFC assay (4). ~ the other hand, those from the thymus, bursa of Fabricius and bone marrow - the so-called central lymphoid organs - showed only marginal activity if at all (1,5). Other studies have shown that despite the lack of antibody-forming capacity of thymocytes, their transfer to newly hatched chicks appeared to awaken precociously the host immune potential (5,6,7). Moreover the enhancement capacity was not restricted to thy313

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mocytes but was exhibited also by spleen cells and blood leucocytes from unprimed juvenile birds (8). Further studies were conducted with the cell transfer model to ascertain (a) if bone marrow cells and bursal cells from unprimed donors possess enhancement capacity, and (b) whether this capacity was susceptible to antigen priming in bone marrow and the bursa. The results indicate that cells with enhancing capacity occur normally at modest levels in the bursa and low levels in the bone marrow of unprimed birds, and that their numbers increase in the bone marrow but not in the bursa after antigen priming.

MATERIALS AND METHODS The chicks and fertile eggs used in the experiments were from the DK/OR White Leghorn chicken lines homozygous f o r the B 13 h apl o type° The erythrocytes used for immunization and antibody assay were obtained from the C3H/ HeJ inbred mouse strain° HA and PFC Assays The limiting dilution method conducted in U-type Microtiter plates was used to estimate the hemagglutinin (HA) titers in the host serum samples. The titers were recorded in Log 2 units. A modified Cunningham slide method was used to estimate the direct-PFC numbers in the host speens (9). Chicken complement, MRBC suspension and lymphoid cell suspensions were prepared in Hanks' balanced salt solution (HBSS) and maintained at ice-water temperature. A mixture composed of 0. 3 ml of a 1:4 dilution of chicken complement, 0.3 ml of lymphoid cell suspension and 0.6 ml of a 4% MRBC suspension was prepared. A 20 microliter drop of the mixture was delivered on to a specially prepared taped slide, and carefully covered with a glass coverslip. The slides were incubated for 30 to 60 minutes in a humidified 38 ° C incubator. Three or more drops from each spleen cell mixture were sampled and after incubation, examined at 100X for PFC numbers. The spleen cells were suspended in NattHerrick diluting fluid (I0) and cell counts of the mononuclear cells were made in a hemacytometer. The PFC counts per 105 mononuclear cells were converted to Log 2 units and the mean values calculated° The mean values in Log 2 units were reconverted to obtain the average PFC numbers. Cell Transfer Assay The bursa was aseptically removed from the donor bird to a dish with RPMI 1640 (with 10% FCS) to prepare cell suspensions. The bursal plicae were cut into small fragments, pressed through a wire screen and the cells dispersed by aspiration through a 20 gauge needle. The bursal cell suspension was washed 3X in RPMI 1640 (with 10% FCS). Bone marrow was flushed from the femurs with sterile Alsever's solution and dispersed by aspiration through a 20 gauge needle attached to a 3 ml plastic syringe. Large cell clumps and tissue fragments were pelleted by light centrifugation and the supernatant cell suspension was washed 3X in fresh Alsever's solution. Cell counts were made and appropriate cell concentrations prepared. About 5 x i0 bursal or bone marrow cells were administered to each host. Depending on the experimental group, MRBC was added to the cell suspension at the time of cell transfer or given to the host at a later time. Six days after exposure to the MRBC the hosts were killed for splenic PFC counts°

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The in vivo culture antibody assay with embryo hosts is used to detect antigen responsive units (ARC) in the donor cell suspension (2). Newly hatched chick hosts are used for the immune enhancement assay (8). The mean and standard errors were calculated for each experimental group and where different groups were compared, the Student t test was used to determine statistical differences between group means.

RESULTS Immune enhancement by bursal cells The immune enhancement capacity of bursal cells from unprimed and MRBCprimed donors were compared° The antigen-primed birds were immunized intravenously with an ml of 1 to % M R B C saline suspension, the concentration used depended on the age. Sixteen unprimed donors, 2 to 9 weeks of age, and fifteen MRBC-primed donors, 2 to 8 weeks of age were tested. MRBC and 5 x i0 ~ bursal cells were administered to each of several assay chicks and six days later the host spleens were examined for PFC numbers. The EC activity levels of bursal cells from unprimed and MRBC-primed donors are compared in Fig. 1. The EC levels in both groups of donors were highly variable, ranging from zero to moderate levels. The group means o f the two groups of donors were not significantly different. The absence of significant difference in the EC activities between the two groups would indicate that the bursal cells are unaffected by MRBC priming.

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Immune enhancement by bone marrow cells In a second experimental series the enhancement capacity of bone marrow cells from unprimed and MRBC-primed donors were compared. Sixteen unprimed birds, 2 to 9 weeks of age, and 15 MRBC-primed bi~ds, 2 to 8 weeks of age, were used as bone marrow donors. MRBC and 5 x 1 0 ° m o n o n u c l e a r bone marrow cells were administered intracardially into each chick host and the spleen examined 6 days later for PFC numbers. Each donor was tested with several assay chicks to obtain representative adoptive FFC values. The EC activities of bone marrow cells from unprimed and MRBC-primed donors are compared in Fig. 2. Immune enhancement was generally low with bone marrow cells from unprimed donors, whereas low to moderate levels of enhancement was observed with cells from immunized donors. A comparison of the group means indicates that the EC levels of the MRBC-primed donors were significantly higher than that of the unprimed group. Apparently MRBC priming elevates the EC numbers in the bone marrow. Moreover the EC level appears to be age-dependent in the primed donor group° Immune enhancement levels in bone marrow as a function of time after immunization Since the EC levels of primed bone marrow donors appear to be affected by MRBC immunization a "response profile" experiment was conducted. In one experiment donor birds, 2 to 3 months of age, were immunized with intravenous injections of 5% MRBC suspension in an ml volume and the bone marrow was assayed for EC activity at 6 hours, l, 2, 4, 5, 6, 7, and 8 days post immunization. A group of unprimed birds served as the pre-immunization control.

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The time profile of EC levels in bone marrow following immunization is shown in Fig. 3. The immune enhancement potential declined from a preimmunization level the first few days but was elevated after the fourth day postimmunization. The preimmunization EC level was higher in these older donors than the unprimed donors shown in Fig. 2. In another experiment, 19 four-to six-week old birds were immunized with MRBC and pairs of birds were tested for bone marrow EC activity on day 2, 4, 6, and 8, and a single bird on day 9. The remaining l0 birds were given a challenge MRBC injection on the 12th day and pairs of donor birds assayed for bone marrow EC activity l, 2, 3, 5, and 7 days later. The EC time profile study of this younger donor group given a primary and challenge doses of MRBC is shown in Fig. 4. The EC level remained at the low preimmunization level the first few days after primary immunization, then rose to a high level by the 4th day and fluctuated thereafter. Following the challenge immunization, the EC level declined from a pre-challenge level to a low level the first few days but then increased to a higher level by the 5th day. Immune activities of embryo hosts grafted with bone marrow cells or bursal cells and exposed to one or two MRBC injections. The following cell transfer experiments were conducted to ascertain if bone marrow or bursal cells were susceptible to MRBC antigen stimulation. Bone marrow cells were transferred into 13 day embryo hosts and MRBC given to the hosts on various days of incubation or at hatching. Six days after the antigen injection the host spleens were examined for PFC numbers. In an alternative experiment, MRBC and bone marrow cells were transferred together into 14 day hosts and then a second MRBC injection given on day 16 of incubation or later. The host spleens were examined for PFC six days after the second antigen exposure. A similar series of experiments were conducted

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with bursal cells. The adoptive immune capacities of cells from unprimed and MRBC-primed donors were compared. Among embryo hosts grafted with bursal cells from unprimed and MRBCprimed donors and exposed to a single MRBC on day 15, 16, 17 or 19 days of incubation or at hatching, the immune activity was clearly apparent in hosts exposed to MRBC at hatching but absent in the younger (embryonic) hosts (see Fig° 5). Similar results were observed in the double immunization series. No significant difference in immune activity was detected between the host group that received MRBC at hatching for the first time and the group exposed to MRBC on day 14 and at hatching. Pre-exposure to MRBC of bursal cells from unprimed donors in the embryo hosts did not increase the adoptive immune activity, whereas those from primed donors did show some elevation as the consequence of double MRBC exposures. In the bone marrow series (Fig. 5) no adoptive immune activity was apparent among embryos grafted with unprimed bone marrow cells and exposed to a single MRBC dose on various days of incubation and at hatching. On the other hand, with cells from primed donors, low to modest levels of adoptive immunity was observed in embryo and chick hosts. Following the double immunization protocol bone marrow from both unprimed and primed donors showed increased responsiveness. The level of immune activity expressed was low in young embryo hosts and appear to increase with older embryo and chick hosts.

DISCUSSION Enhancer cells (EC) are defined operationally as immunosupportive cells, which although incapable of adoptive immunity in embryo hosts, are able to enhance the immune responsiveness of chick hosts. It was found that the EC activity was low in bone marrow of unprimed birds but elevated in that of MRBC-primed donors. Low to modest EC levels were found in the bursae of both unprimed and primed donors. Following immunization the EC level of bone

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marrow increased from a low level at I to 2 days to a high level by day 6 to 8. When bone marrow cells from unprimed and primed donors and bursal cells from primed donors were transferred to embryo hosts and exposed to two doses of MRBC at least three days apart, adoptive immunity was observed in the embryo recipients. Bursal cells from unprimed donors, however, were unresponsive. The apparent deficiency in adoptive immune capacity of bursal and bone marrow cells and the unresponsiveness of bursal cells to antigen stimulation was not unexpected since cells of the central lymphoid organs are assumed to be unreactive to antigens (11,12). Although earlier studies might indicate the absence of antigen responsive cells in the bursa (13,14),others have reported the presence of immunocompetent B cells in the bursa which are capable of at least local antibody production (15-17). However in our experience FFC formation was not elicited in the bursa with intravenous MRBC immunization. The capacity of bursal cells to enhance antibody formation in chick hosts but not in embryo hosts resembles the behavior of transplanted thymocytes (8). This behavior of thymocytes was attributed to T W precursors which are more abundant than B cell precursors in the thymus. AIthough the predominant immunocyte precursors in the bursa is of the B cell line, small numbers of T cells and their precursors have been reported in this organ (18,19) that could account for the weak immune enhancement effect. However, the possible contribution of the few mature B cells in the bursa (20) toward the formation of I~FC in the embryo hosts following their transfer cannot be excluded. Cell transfer studies by others have shown that bursal stem cells

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and postbursal cells in this organ can restore the immune capacity of cyclophosphamide-induced immunodeficient chicks (21). Because of the heterogeneous nature of the bursal cell suspensions used in the present study, it is possible that the immune enhancement observed with these cells is the result of the combined effect of TH cells and B-cell precursor activities.

Whereas bursal cells appear unreactive to antigen stimulation within the embryo host system, thymocytes as reported earlier (8) and bone marrow cells were responsive. Moreover, enhancer cells were found in varying numbers in all these tissues. The elevated EC levels in MRBC-primed donor bone marrow reflects an increase in the population of immunocompetent cells as the consequence of antigen stimulation. The increase could be accounted for by (i) clonal expansion of MRBC-sensitive cells native to bone marrow or (2) increased recruitment of blood-borne immigrant immunocytes of extramedullary origin. Bone marrow is the definitive site of B cell origin and contains the early precursors of antibody-forming cells (22). Pre-PFC have been identified in the mouse bone marrow (23) and Ig-bearing (24,25) and Ig-secreting cells (26) have been reported also in the chicken bone marrow. The antibody forming cells of the bone marrow are believed to originate ultimately from the bursa (25,27,28), but the situation in chickens is still unsettled. There is little direct evidence from our experiments that would indicate that mature B cells occur in sufficient n u m b e r s i n the bone marrow of juvenile birds to account for the high immune activity observed. Moreover, the inability of transferred bone marrow cells from unprimed donors to respond to single immunization in embryo hosts suggests the relative paucity of ARC. The marginal adoptive immune capacity and the modest enhancement effect of bone marrow cells from antigen-primed donors is explainable by an influx of blood borne precursors. The resemblance of the bone marrow EC time profile to the immune response profile could just as well reflect the production of immunocyte precursors at peripheral sites, their release into the circulation and subsequent migration to the bone marrow. Immunocyte precursors exist in the blood stream of unimmunized birds (8) and their numbers increase dramatically soon after immunization (i). It is difficult therefore to exclude migrant cell contamination from the peripheral blood. The apparent elevation of EC levels in bone marrow following immunization is not in itself sufficient evidence for reactivity of intrinsic bone marrow cells and their subsequent population expansion. Related studies in the mouse emphasize the importance of peripheral tissues as the source of bone marrow immunocompetent cells. Parabiosis and splenectomy experiments indicate that the bone marrow immunocyte populations are predominantly migrant cells (29, 30) and include not only B cells but long-lived memory T and B cells as well (30,31).

Artificial immunization with MRBC is associated with increase in immunosupportive cells in the thymus (8), increased production of ARC aud EC in the spleen and possibly in the peripheral blood (1,8), their release into the blood stream and their transportation to other sites, including the bone marrow. Although the effect of antigen on the bursa is less direct, it appears that even this organ possesses cells with capacity for immune responsiveness (16,17). The kinetics of lymphoid cell migration from the central lymphoid tissues to the peripheral sites during the perinatal period and early juvenile period are yet incompletely understood, but it is conceivable that natural antigen encounters accelerate the process.

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ACKNOWLEDGEMH~TS

Research supported in part by a grant-in-aid from the Oklahoma University Research Council and from the USPHS Biomedical Research Support Grant to the University of Oklahoma.

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2.

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Received : May, 1982 Accepted : October, 1982