Chicken Spleen Cells Research Articles

Antibody synthesis stimulation by vitamin A in chickens

The effect of vitamin A on the immune response of chickens on primary and secondary immunization has been studied. It is demonstrated that the antibody content in chickens depends on the dose of vitamin A in the diet. In chickens fed a high dose of vitamin A in the diet the antibody content in the serum is 2 to 5 times as high as in chickens which have not been given vitamin A. An additional dose of vitamin A administered perorally any day following reimmunization increases the content of antibodies; the effect is most pronounced when vitamin A is given on the third day after reimmunization. In the period of maximal synthesis of antibodies (during the fourth day after reimmunization) labelled retinol is incorporated into the spleen of reimmunized chickens 13 times as actively as in the spleen of control chickens. In the in vitro experiments with antibody synthesis by spleen cells it has been demonstrated that addition of retinyl palmitate to the incubation medium enhances antibody synthesis during incubation. Addition of retinyl palmitate to the spleen cells of vitamin A-deficient chickens restores the synthesis up to the level observed in the control cells. The suggestion is discussed that the effect of vitamin A on the immune process is realized at the level of immunoglobulin synthesis.

Effects of Lymphocytes from Marek's Disease-Infected Chickens on Mitogen Responses of Syngeneic Normal Chicken Spleen Cells

PHA responses have been measured in lymphoid cell cultures prepared by mixing normal chicken spleen cells with spleen or thymus cells from syngeneic chickens infected with the oncogenic herpesvirus MDV. Results of these studies may be summarized as follows: 1) spleen cells from MDV-infected chickens with visceral lymphomas inhibit the PHA response of normal spleen cells possibly by release of soluble inhibitory factors in response to the mitogen; 2) lymphoid cells from asymptomatic MDV-infected chickens, although hyporeactive themselves to PHA, can have a stimulatory effect on PHA responses of normal spleen cells in mixed cultures; 3) spleen cells from MDV-infected chickens, effectively protected from viral oncogenesis by HVT vaccination, show normal reactivity to PHA in spearate cultures and may react in mixed cultures like normal lymphocytes, with neither a pronounced stimulatory nor inhibitory effect on the PHA response of normal spleen cells.

EA rosette forming lymphoid cells: Distribution as to cell types and organs from chickens of different developmental stages

The interaction of chicken spleen cells with sheep erythrocytes coated with chicken antibody (EA complexes) was studied using a rosette assay. The results reported in this paper indicate that subpopulations of chicken lymphocytes, monocytes, and heterophils have a receptor for EA. The formation of rosettes between chicken lymphoid cells and sheep erythrocytes (SRBC) was dependent upon the concentration of antibody used to sensitize the SRBC. In a developmental study of rosette-forming lymphocytes (RFL), the bursa was the first site of appearance of large numbers of RFL. The percentage of RFL in the bursa reached a peak at 17 days of embryonic life, and declined to a low by hatching. The percentage of RFL in the spleen, however, began to increase at the time of hatching and by 6 weeks of age the spleen far surpassed the bursa in percentage RFL. At no age were significant numbers of RFL detected in the thymus.

Antiserum specific for reticulin of the bursa of fabricius.

Antiserum produced by immunizing rabbits with embryonic chicken spleen cells showed, after appropriate absorption with cell suspensions, specificity for fetal spleen cells in membrane immunofluorescence tests. However, when the original antiserum was absorbed with tissue homogenates and tested on cryostat tissue sections, it reacted specifically with the reticular framework which separates the cortical and medullary lymphocytes in the follicles of the adult bursa of Fabricius. It is suggested that the reticulin may influence bursal lymphoid differentiation.

Antibody synthesis by chicken spleen cells in vitro. I. Requirements of B cells at various stages after immunization for T cells, macrophages and antigen.

AbstractThe requirements for T cells, macrophages and antigen during the induction of in vitro antibody responses were ascertained with chicken spleen cells obtained at various times after immunization with sheep red blood cells (SRBC). The IgM plaque‐forming cell (PFC) response was T cell independent exclusively in cultures initiated 3 days after priming, but macrophage dependent at all time intervals tested. In cultures started 4 to 10 days after priming the IgG response was both T cell and macrophage independent and PFC numbers remained at a high plateau level throughout the culture period. In contrast, IgG responses initiated more than 15 days after priming showed a reversal to complete T cell and macrophage dependence and were characterized by a sharp increase in PFC numbers between days 2 and 4 of culture. Formaldehyde‐fixed SRBC were immunogenic for IgG PFC 4 to 10 days after priming but failed to stimulate later IgG memory and all IgM responses. Contrasting antigen dose requirements for IgM and IgG responses were found in cultures initiated at various periods after priming.The results suggest that direct contact with fixed antigen was sufficient to maintain IgG antibody synthesizing PFC in vitro, while native antigen and cell co‐operation were required for late secondary IgG and all IgM responses. These results are interpreted in terms of separate pathways of differentiation for IgM and IgG antibody‐producing cells. A distinct 3rd day stage of T cell‐independent but macrophage‐dependent responsiveness for both classes of antibody was also defined.

In vitro response of chicken spleen cells to sheep red blood cells

A modification of the Marbrook system for obtaining both primary and anamnestic responses from chicken spleen cells in vitro is described. The patterns of responsiveness of chicken spleen cell cultures resembled those previously described for the mouse. However, the requirement for normal chicken serum (NCS) to support the response could not be replaced by foetal calf serum (FCS). Cell survival, response kinetics, antigen dose requirements, antibody class, and temporal development of memory are described. The requirement for cooperation of B and T cells in the SRBC response was demonstrated by the use of specific anti-T and anti-B sera.

Primary antibody response by chicken spleen cells in vitro.

An in vitro method that permits the primary antibody response of chicken spleen cells to sheep erythrocytes to occur is described, and the characteristic of the antibody response are given. Significant but variable plaque‐forming cell (PFC) responses were obtained—that is, some animals showed no or a very weak response In these the addition of small numbers of allogeneic spleen cells much improved the PFC responses. The characteristics of this allogeneic stimulation are described. The presented culture method should facilitate studies of cell interactions in the primary antibody response of the chicken and in the development of the antibody‐forming cells.

Selective proliferation of T cells in the mixed lymphocyte interaction

The contribution of avian thymus-derived (T) and bursa-derived (B 2) cells to the proliferating cell population in mixed lymphocyte cultures (MLC) was evaluated. When spleen cells of chickens containing chromosomally marked T and B 2 cells were stimulated in a one-way MLC by mitomycin C blocked allogeneic spleen cells, only T cells proliferated during a 4–9 day culture period. No evidence for significant recruitment of B 2 cells, expressed as proliferation of B 2 cells, was found. The initial viability and proliferative potential of B 2 cells was shown by a substantial and selective B 2 cell response to anti-immunoglobulin serum.

Direct evidence for the response of B and T cells to pokeweed mitogen

Chicken spleen cells containing chromosomally marked thymus derived (T) and bursa-derived (B 2) cells were evaluated for their ability to respond to pokeweed mitogen (PWM), concanavalin A (Con A), and to anti-immunoglobulin serum during a 4-day culture period. The results indicate that soluble PWM induces a proliferative response of B 2 cells in addition to a predominant T cell response. The PWM-induced B 2 cell proliferative response was clearly detected only at 4 days after culture initiation. Soluble Con A did not induce detectable proliferation of B 2 cells and stimulated T cells exclusively. In contrast, anti-immunoglobulin serum was a specific stimulant for B 2 cells under the culture conditions used.

The Production of a Monocyte Chemotactic Factor by Agammaglobulinemic Chicken Spleen Cells

Abstract The classification of lymphocytes into bursal derived (B)2 and thymic derived (T) subpopulations has been a significant advance in our understanding of lymphocyte biology. A variety of immune functions have been attributed to each of these subpopulations, while other functions depend on a cooperation between both cell types (1). It has been suggested that lymphokines, the biologically active substances produced in vitro by activated lymphocytes (2, 3) are produced by T lymphocytes (2). However, little definitive experimental evidence to substantiate this conclusion has been reported. The agammaglobulinemic (aγ) chicken has been shown to be largely depleted of B cells (4, 5) and represents an excellent model in which to investigate the contributions of B and T cells to various aspects of the immune response (6). In the present investigation we have adapted a recently described assay of mononuclear leukocyte (MNL) chemotaxis (7) to study lymphokine production by mitogen-stimulated normal and aγ chicken spleen cells.

Inhibition of Antigen-Stimulated in Vitro Proliferation of Thymic Dependent Chicken Spleen Cells by Specific Antibody

Abstract The ability of passively administered antibody to inhibit active immune responses suggests that antibody may be an important element in normal immunoregulation. This report describes studies of in vitro lymphocyte transformation in which cells from agammaglobulinemia bursectomized-irradiated (Bx-X) and rabbit anti-chicken lymphocyte serum (ALS)-treated chickens were used to study the effects of specific antibody on the in vitro proliferative responses of thymus dependent (T) and bursal dependent (B) lymphocytes. Intact chickens immunized with keyhole limpet hemocyanin (KLH) and Brucella abortus (BA) developed positive in vitro proliferative responses to these antigens and these responses were specifically inhibited when the appropriate antibody was added to the cultures. Low level “natural” or background proliferative responses were observed in unimmunized chickens in response to Brucella, and these responses could not be inhibited by specific anti-Brucella (anti-BA) antibody. Agammaglobulinemic (Bx-X) chickens immunized with both KLH and Brucella responded in vitro only to KLH. This response was also inhibitable by specific anti-KLH antibody, indicating that proliferative responses of T cells can be suppressed by specific antibody. Because spleen cells from immunized Bx-X birds failed to respond to Brucella, the in vitro proliferative response to this antigen is probably a B lymphocyte dependent process. This was investigated further by using spleens from immune ALS-treated chickens. These cells failed to respond to either antigen in vitro. When these ALS spleen cells were mixed with immune Bx-X spleen cells, the in vitro proliferative response to BA was reconstituted, demonstrating synergy between the two populations of cells. However, since both T and B cells are needed for the proliferative response to BA, we cannot determine which lymphoid cell was inhibited by anti-BA.

Interaction of embryonic chicken spleen cells and adult allogeneic leucocytes.

SummaryLeucocytes from inbred AA chickens produced pocks when inoculated onto the chorioallantoic membrane of non‐inbred embryos (direct pock formation), but produced very few or no pocks on the chorioallantoic membrane of syngeneic embryos. Pock formation was observed when AA leucocytes were mixed with non‐inbred embryonic spleen cells before being assayed on the chorioallantoic membrane of recipient embryos that were unresponsive to either cell population alone (indirect pock formation).Indirect pock formation was found to be more radio‐resistant than direct pock formation and doses of Mitomycin‐C that inhibited direct pock formation by AA leucocytes did not inhibit the capacity of these cells to induce indirect pocks. Indirect pock formation was inhibited when non‐inbred embryonic spleen cells were treated with Mitomycin‐C prior to mixing with AA leucocytes. It is suggested that indirect pock formation does not result from the stimulation of adult leucocytes by antigen presented on the surface of allogeneic embryonic cells. Rather, this response is a manifestation of the direct stimulation of embryonic spleen cells by adult allogeneic leucocytes.

IN VITRO STUDIES OF CHICKEN LYMPHOID CELLS

A tissue culture technique for the study of the mixed leucocyte reaction (MLR) occurring between allogeneic chicken spleen cells was developed. Some features of the one‐way MLR, using mitomycin C‐treated spleen cells as stimulators, and the two‐way MLR were characterized. Cells forming antibody to histocompatibility or other antigens common to lymphoid cells and erythrocytes could not be detected in the MLR. Spleen cells from hypogamma‐globulinaemic bursectomized‐irradiated chickens reacted normally both in the two‐way and one‐way MLR. It is suggested that the MLR occurs between thymus‐dependent lymphoid cells or between such cells and macrophages. Bursa‐dependent lymphoid cells appear to play a non‐essential role, if any, in the MLR.

Activation of Non Competent Embryonic Chicken Spleen and Bone Marrow Cells by Thymus and Bursa Fabricii Extracts

Previous studies have shown both thymus and bursa cells to activate a pre-existing competence in adult lymphoid cells, probably by means of a humoral factor, Klopstock and Sharon (1965), Miller (1965), Small and Trainin (1967).Rather than using intact cells as inducers we were interested in finding out whether an extract of thymus or bursa cells could be shown to activate immunologically immature cells.For this purpose, two sorts of inactive cells, Simonsen (1965), Burnet and Burnet (1960) were chosen:1. chick embryo bone marrow cells2. chick embryo lymphoid cellsTo measure the immunologic competence of these cells, following their expected activation by extract from bursa and/or thymus cells, we decided to use the Simonsen CAM phenomenon, Cock and Simonsen (1958). The Simonsen phenomenon, as is known, entails the production of focal lesions on the chorio-allantoic membrane (CAM) of chick embryos upon placement on the membrane of immunologically competent…

Molecular hybridization of RNA from chicken spleen cells after immunization.

Es wird mit neuer Technik gezeigt, dass nach Immunisierung von Huhnern in der Milz neue Ribonukleinsauren erscheinen.

Effect of thalidomide on the graft versus host reaction.

LYMPHOID cells from a highly inbred parental strain of rat injected into F1 hybrids derived from that strain and another inbred strain provoke a reaction, characterized among other manifestations by a hyperplasia of the recipients' spleen and other lymphoid tissues1. A striking splenic enlargement can also be produced in the chick embryo by intravenous injection of adult chicken spleen cells, or peripheral white blood cells2. The splenomegaly in both instances is due to an attack by the inoculated immunologically competent cells directed against a host incapable of reacting against them3–5. The splenomegaly is part of a more generalized graft versus host (GVH) reaction and reduction of the degree of splenomegaly is therefore a measure of the inhibition of the GVH reaction6,7.

An analysis of host and donor contributions to splenic enlargement in chick embryos inoculated with adult chicken spleen cells

The phenomenon of embryonic splenomegaly in the chick embryo has been investigated quantitatively to obtain information that would further elucidate the nature of the splenic response to grafted competent tissue. The effects of varying the donor cell number, host embryo age, and X-irradiation exposures of donor spleen cells and host embryos on splenic enlargement were investigated and the cellular dynamics of the splenic enlargement was analyzed. Following are listed the salient findings of the investigations: 1. 1. Within the range of embryo ages studied, a fourfold increase in donor cell number resulted in an approximate doubling of the plateau level of spleen weight. The relationship between donor cell number and splenic enlargement is suggestive of an antigen dosage effect, where either donor cells or host embryo is considered as “antigen.” 2. 2. The maximum splenic enlargement resulting from injection of the maximum permissible number of donor cells varied with the age of the recipient embryo, being minimal in 8- to 10-day-old hosts, maximal in 12- to 13-day-old hosts, and declining in older embryos. 3. 3. Irradiation of either donor spleen cells or recipient embryos prior to inoculation is equally effective in reducing splenic enlargement, and the decrease was radiation-dose dependent. 4. 4. The predominant spleen cell type present during peak splenic enlargement is the blast cell, and it increases in number at a faster rate than do other cell types during enlargement of the spleen. 5. 5. A calculation based on the number of H 3-TdR-labeled donor spleen cells which settle in the spleen initially and proliferate indicates that the number is too small to account for the splenic enlargement. The activation of the reservoir of host proliferative cells, however, can easily account for the observed enlargement. The experimental findings reported in this paper indicate that both host embryos and grafted adult spleen cells participate in the splenic enlargement. The possibility of a host-immune response toward grafted donor spleen cells has been discussed.

INOSITOL AND THE PROTECTION OF CELLS. II. THE RELEASE OF NUCLEIC ACIDS FROM NORMAL AND MALIGNANT CELLS.

Various normal mouse or chicken cell types or Ehrlich ascites cells were incubated for periods of up to 3 hours in either Hanks' balanced salt solution (BSS) or 5% inositol solution. Cells incubated in inositol invariably showed better viability as judged by erythrosin staining than those incubated in BSS. Ehrlich cells incubated in BSS showed a reduced ability to take up oxygen after 90 minutes incubation while those in inositol did not. Mouse embryo and chick embryo cells were able to grow in tissue culture even after prolonged treatment with 5% inositol.Maintenance of cell viability was accompanied by a greater loss of RNA from the cell in inositol compared with BSS. This loss appeared to be relatively greater from normal cells than from Ehrlich cells, and in the case of chicken spleen the loss of RNA after 2 hours incubation in inositol was 10 or more times greater than in BSS. Purification and base composition data are presented for inositol-extracted RNA from normal chicken spleen cells, Ehrlich cells, and a subline of Ehrlich. Differences in base composition are apparent for each cell type. Some implications of these findings are discussed.

TRANSFER TO NEONATAL CHICKENS OF THE PRIMARY ANTIBODY RESPONSE INITIATED IN VITRO.

THERE are numerous reports on the formation of antibody by neonatal animals which had been injected with homologous spleen and lymph node cells mixed in vitro with bacterial antigens1–3. Transfer to neonatal recipients of a primary response to serum protein antigens under similar conditions has often met with failure or with poor antibody responses3. The latter has been attributed either to the unsuitability of the neonatal environment for the cells4,5 or to the possibility that soluble proteins have a longer induction period, and as a consequence the homograft reaction results in damage of the cells before antibody is demonstrated6. To obviate this, Holub and Řiha utilized diffusion chambers which protected the transferred adult cells from the rejection mechanism. The present report summarizes further attempts to initiate an antibody response by in vitro mixing of chicken spleen cells with either bovine serum albumin (BSA) or with BSA–anti-BSA complexes, and subsequent transfer to neonatal chickens.

Immunological Attack by Adult Cells in the Developing Chick Embryo: Influence of the Vascularity of the Host Spleen and of Homograft Rejection by the Embryo on Splenomegaly

ABSTRACT Adult chicken spleen was implanted on to the chorio-allantoic membrane of chick embryos at 8,9,11,13 and 15 days of incubation. The initial manifestation of graft against the host attack by these adult cells was an increase of host spleen weight (splenomegaly). No splenomegaly appeared in the embryonic hosts before 15 days of incubation. The appearance of splenomegaly roughly coincided with the appearance of reticulum cell foci in the chimaeric spleens. Varying latent periods between the time of introduction of donor cells and the first appearance of increased spleen weights necessitated comparison of the host spleen weights 3 days after the onset of splenomegaly. This showed that implantation at 13 days produced the greatest splenomegaly. Only very slight splenomegaly was obtained after implantation of spleen at 15 days. A possible homograft rejection by the host and poor ‘take’ of the splenic implant on the chorio-allantoic membrane are both thought to be responsible. Intravenous injection of adult chicken spleen cells into embryos at 6, 8, 11 and 13 days of incubation again produced no splenomegaly before 15 days of incubation. Comparison of the host spleen weights 3 days after onset of splenomegaly showed that injection at 13–15 days gave the greatest splenomegaly. Injection before this time gave very slight splenomegaly and injections after 13 days produced a decreasing degree of splenomegaly with increasing age of embryo injected. Increasing the number of adult spleen cells at 15 days or giving large doses of blood at 8, 9, 11, 15 and 17 days of incubation and in the 1-and 2-day-old chick failed to increase the splenomegaly greatly or markedly to alter the above pattern. When 1-day-old chicks were injected with allogenic blood only slight splenomegaly occurred and when 2-day-old chicks were injected there was no splenomegaly 6 days later. The suitability of the chick embryo as an environment for the proliferation of immunologically competent cells is discussed. The reduction in splenomegaly when embryos older than 13-15 days are injected is discussed in relation to the appearance from this time of a weak homograft response by the embryo and to the greater difficulty of induction of both splenomegaly and tolerance to skin grafts soon after hatching. It is proposed that very small numbers of immunologically competent cells appear in the embryo soon after 15 days and progressively increase from this time until onset of competence of the host at 10 days post-hatching. Possible effects of such cell growth on the degree of induction of tolerance are discussed.