Restorative effect of neurotropin on maturation of bone marrow cells to IL-2-producing T-cells in aging BALB/c mice.

In a previous paper, we have demonstrated that Neurotropin, a non-protein extract isolated from the inflamed skin of rabbits inoculated with vaccinia virus, restores decreasing immune responses through the recovery of interleukin-2 (IL-2) production in aging BALB/c mice. To clarify the mechanism by which Neurotropin restores IL-2 production, its effect on the recruitment of IL-2-producing T-cells from bone marrow cells was examined using syngenic radiation bone marrow chimeras. Two fundamental lesions in recruiting IL-2-producing T-cells in aging BALB/c mice were demonstrated: (1) a drastic decline of the maturation of bone marrow cells to IL-2-producing T-cells as demonstrated by old----young chimeras; and (2) an environment unable to support bone marrow cell differentiation to IL-2-producing T-cells by young----old chimeras. Neurotropin clearly restored the maturation of bone marrow cells to IL-2-producing T-cells when administered from 13 to 16 month-old mice, whereas the non-complementing environment was not normalized with Neurotropin administration. These results suggest that Neurotropin administration restores IL-2 production through the recovery of the maturation of bone marrow cells to IL-2-producing T-cells, resulting in restoration of in vivo T-cell immune response in aging BALB/c mice.     

Immunomodulatory effects of neurotropin through the recovery of interleukin-2 production in autoimmune-prone (NZB/NZW) F1 mice

The immunomodulatory effects of Neurotropin, a substance extracted from inflammatory skin of rabbits inoculated with vaccinia virus, were assessed in autoimmune-prone (NZB/NZW) F1 (B/W F1) mice. The concanavalin A (Con A)-induced proliferative response of spleen cells was markedly decreased in aged B/W F1 mice as compared with young B/W F1 mice. Neurotropin, when administered i.p. to aged B/W F1 mice, significantly increased the Con A-induced proliferative response. In aged B/W F1 mice, interleukin-2 (IL-2) production by Con A-stimulated spleen cells was severely impaired and IL-2 responsiveness of Con A-activated spleen cells was partially decreased in comparison with young B/W F1 mice. Neurotropin, administered to the aged B/W F1 mice, restored IL-2 production by Con A-stimulated spleen cells to the level of young B/W F1 mice. Furthermore, Neurotropin completely restored the IL-2 responsiveness of Con A-activated spleen cells from aged B/W F1 mice. To test whether Neurotropin exerts its immunoregulatory activities in B/W F1 mice by restoring IL-2 production, we directly examined the effect of recombinant IL-2 on the immune functions of spleen cells in vitro. Recombinant IL-2 markedly enhanced Con A-induced proliferative response of aged B/W F1 mice. Furthermore, the suppressive activity of spleen cells which had been activated by Con A in the presence of rIL-2 was significantly increased. These results indicate that some immunoregulatory functions of aged B/W F1 mice can be corrected by IL-2 and suggest that Neurotropin restores immunoregulatory activity in B/W F1 mice by the recovery of IL-2 production.     

Effects of a novel anti-inflammatory retinoid-like 2,4,6,8-nonatetraenoic acid on the immunological changes associated with adjuvant-induced arthritis

Proliferative responses to the T-cell mitogen, Con A, were markedly suppressed in spleen cells isolated from rats 12-16 days following induction of adjuvant-induced arthritis (AA). These responses were only partially restored following removal of plastic-adherent cells (AC-depleted). Prophylactic treatment of AA rats with a novel anti-inflammatory retinoid-like 2,4,6,8-nonatetraenoic acid, Ro 23-6457, increased mitogen-induced proliferative responses in spleen cells, particularly in AC-depleted cultures. Treatment of AA rats with Ro 23-6457 significantly increased Con A-induced IL-2 production by both unseparated and AC-depleted spleen cells. Although exogenous IL-2 did not restore proliferative responses to Con A-stimulated spleen cells from vehicle-treated AA rats, responses in AC-depleted cells from Ro 23-6457-treated AA rats were further enhanced by the addition of IL-2. Following stimulation with LPS, supernatants from cultures of adherent spleen cells isolated from AA rats contained more IL-1 (expressed as units/ml) than cultures from normal rats. Treatment of AA rats with a high dose of Ro 23-6457 normalised IL-1 levels in these cultures. Treatment of normal rats with Ro 23-6457 had no significant effects on any parameter tested. These data suggest that Ro 23-6457's modulation of certain disease-associated alterations in immune function in AA rats may contribute to its anti-inflammatory activity.     

Immunoregulatory cells in aging mice. I. Concanavalin A-induced and naturally occurring suppressor cells

The status of concanavalin A (Con A)-triggered suppressor cells in aging mice was assessed in relation to untreated cells. Spleen cells of (C3H/ebJ X C57BL/6J)F1 and (BALB/cJ X C57BL/6J)F1 aging (20-41 months' old) and young (2-3 months' old) mice were triggered with Con A and examined for their capacity to suppress lymphocyte proliferative responses elicited by mitogens (Con A and phytohemagglutinin), by mixed lymphocyte reactions or by human gamma-globulin. Con A-triggered cells of the aging mice were found to be less efficient suppressors than those of the young, yet the aging mouse spleen cells exerted a more potent suppressive effect than the young, a priori, without pretreatment. Hence, the immune system in aging is characterized by a change in suppressor cell types; the Con A-triggered suppressors diminish and the naturally occurring, untreated ones become abundant.     

In vivo administration of cytokine in allogeneic bone marrow chimeras.

When we analyzed the in vivo efficacy of cytokine administration in murine allogeneic bone marrow chimeras, mitogen-induced responses to ConA, PHA, LPS, or PWM were increased by the in vivo administration of human recombinant granulocyte colony-stimulating factor (rG-CSF), human recombinant interleukin 2 (rIL-2), or WEHI-3B conditioned medium (CM). Furthermore, we found increased alloreactive mixed lymphocyte reactions (MLRs) against donor and/or host type alloantigens in spleen cells from (BALB/c----C3H/He) chimeras, although cytotoxic activity against BALB/c or C3H/He target cells was not detected in spleen cells from these chimeras. Since no significant increase of T cells or Ia positive cells was observed, some functional activation, rather than changes in the cell count, appeared to relate to increase immunoreactivity. An increased IL-2 production in spleen cells from chimeras injected with cytokine was observed shortly after the cessation of cytokine administration. Thereafter, an IL-2 production in these chimeras decreased around 45 days after bone marrow transplantation and then recovered nearly to the control level. An increased IL-2 responsiveness was also observed in spleen cells from these chimeras. These findings suggest that the in vivo administration of rG-CSF as well as rIL-2 or WEHI-3B CM (IL-3) can modulate the immunoreactivity in chimeras via the network of immune systems.     

Early IFN-gamma production is related to the presence of interleukin (IL)-18 and the absence of IL-13 in experimental Trypanosoma cruzi infections

Gamma-interferon (IFN-gamma) production, the hallmark of the Th1 immune response, has been shown to play a central role in the resistance to Trypanosoma cruzi infections, in particular when produced in the very early acute infection. BALB/c mice infected with T. cruzi, Tulahuén strain, reach high parasitemias during the acute phase, and their spleen cells release IFN-gamma in the second week of the infection, while those of the resistant C3H strain produce the cytokine earlier, at 2 days post-infection (pi). We studied in the spleen cells supernatants of infected BALB/c and C3H mice, the spontaneous production of cytokines involved in the induction, interleukin (IL)-18 and IL-12 p70, as well as in the downregulation, IL-13 and IL-10, of the Th1 immune response. We found that, at 2 days pi, only C3H mice produced IL-18, while IL-12 p70 was detected in both mouse strains. Moreover, at this time pi splenocytes from BALB/c mice spontaneously produced high amounts of IL-13. At 14 days pi, despite the increased levels of IL-13 and IL-10 detected in C3H mice, they still showed high concentrations of IL-18 and IL-12 p70. In contrast, spleen cells from BALB/c mice did not secrete IL-18, IL-12 p70 and IL-13 at this time pi, but produced higher amounts of IL-10 than C3H mice. Non of these cytokines was found increased in the cell supernatants of chronically infected mice. The addition of lipopolysaccharide (LPS) or Concanavalin A (Con A) to the cell cultures did not enhance the production of IL-18 and IL-12 at the time points tested. On the other hand, at 21 days pi, when parasitemia peaked, an inhibition of both the LPS induced IL-10 release and the IL-13 production upon Con A stimulation was observed in C3H, but not in BALB/c mice. We did not find an increase of IL-18, IL-10, or IL-12 p70 in the serum of the infected mice, despite the high seric IL-12 p40 concentrations reached during the infection. The data show that the different kinetics of the production of these cytokines in the spleen of both mouse strains could have a key role in the in vivo regulation of IFN-gamma production. In these experimental models, early IFN-gamma release and thus resistance to T. cruzi infection, could be related to the combined effect of both IL-18 and IL-12p70 in the absence of IL-13.     

Effects of granulocyte-macrophage colony stimulating factor (GM-CSF) in vivo on cytokine production and proliferation by spleen cells

GM-CSF is a potent stimulator of haematopoietic cells as well as some functions of granulocytes and macrophages. GM-CSF has many clinical uses; however, little is known about the effects of GM-CSF treatment in vivo on the responses of tissue lymphocytes in terms of secretion of Th-1 and Th-2 cytokines. We investigated this issue by measuring the responses of spleen cells from mice 24 h after treatment i.p. with saline or rmGM-CSF. GM-CSF at 16.7-50.0 microg/kg significantly increased (P < 0.01) spleen cellularity 2-2.5-fold and enhanced proliferative responses of non-stimulated (no mitogen) as well as concanavalin A (Con A)-stimulated spleen cells. Secretion of IFN-gamma by Con A (2.5 microg/ml)-stimulated spleen cells was significantly (P < 0.01) increased from 1.8 microg/ml by control spleen cells to 5.2 microg/ml by GM-CSF spleen cells. IL-10 production was greater (0.25 microg/ml, P < 0.05) by Con A-stimulated spleen cells from GM-CSF-treated mice compared to control spleen cells (0.06 microg/ml). By contrast, there were no significant differences in IL-4 production by Con A-stimulated spleen cells from the different groups. These results show that GM-CSF treatment increases spleen cellularity and primes lymphocytes for enhanced responses. The enhanced production of Th-1 cytokines by primed lymphocytes may partially explain the beneficial role of in vivo administration of GM-CSF in several clinical situations.     

Proliferative and cytotoxic immune functions in aging mice. III. Exogenous interleukin-2 rich supernatant only partially restores alloreactivity in vitro

The ability of exogenous interleukin-2 (IL-2) rich supernatant to restore the defective T cell mediated immune functions of spleen cells from aged C57BL/6 mice was analyzed. Addition of IL-2 rich supernatant to allogeneic mixed lymphocyte cultures (MLC) resulted in an increase in the proliferative response of spleen cells from both young and old mice. The MLC response of cells from old mice was, however, not restored to the level of proliferation seen with splenocytes from young animals. In studying the generation of specific T cell suppressor function, it was found that IL-2 rich supernatant enhanced this function only for spleen cells from those aged animals which demonstrated a defective response in its absence. The response of these mice was thereby restored to the normal level. The response of cells from young control animals and aged mice with normal suppressor activity was not affected by the addition of IL-2 rich supernatant. We conclude that decreased IL-2 production constitutes a functionally important aspect, but is by no means the only defect in the immune response of aged mice. The results also suggest that responsiveness to IL-2 is less affected by age than lymphokine production.     

犬蛔虫（Toxocaracanis）对感染鼠的细胞免疫和IL－1、IL－2免疫调节作用的影响

应用犬蛔虫（Ｔ．ｃａｒｎｉｓ）的感染性虫卵口饲感染Ｃ３Ｈ／ＨｅＮ鼠，体外培养后观察脾细胞的免疫学变化。感染第１－４周的脾细胞用ＣｏｎＡ刺激，Ｔ细胞的增殖反应，ＩＬ－２的诱生均明显地受到抑制，而且感染鼠脾细胞抑制了正常鼠脾细胞对ＣｏｎＡ的反应。感染鼠的脾细胞经ＳｅｐｈａｄｅｘＧ－１０过柱后，Ｔ细胞则不显示被抑制作用。表明影响Ｔ细胞被抑制作用的是感染鼠的巨噬细胞。相反，感染第１－４周的鼠所产生的ＩｇＧ和ＩｇＭ等抗体的Ｂ细胞活性增强；用ＬＰＳ刺激巨噬细胞诱生的白介素（ＩＬ）ＩＬ－１也增多。     

Effect of hydroxyurea on the concanavalin-A proliferative response of Trypanosoma cruzi infected mice

Mice infected with Trypanosoma cruzi develop immunosuppression with a deficient production of interleukin-2 (IL-2). In this situation the deficient concanavalin A (Con A) T-cell response is not corrected by addition of exogenous IL-2. Here we show that elimination of cycling cells by treatment of infected mice with hydroxyurea (HU) fully restored the ability of spleen cells to respond to IL-2. Further, capacity for IL-2 production was restored to HU treated infected mice, but not as completely as the response to IL-2.     

Hexachlorobenzene treatment increases the number of splenic B-1-like cells and serum autoantibody levels in the rat.

The immune responsiveness of cats naturally or experimentally infected with feline immunodeficiency virus (FIV) was studied. Peripheral blood mononuclear cells (PBMC) from naturally infected, symptomatic animals displayed depressed proliferation and interleukin-2 (IL-2) production in response to mitogens, which was accompanied by a significant increase in IL-1, IL-6 and tumour necrosis factor (TNF) production. Longitudinal studies were performed over a period of 4 years in experimentally infected animals. The responses of cells from these cats to concanavalin A (Con A) were consistently less than those from uninfected cats throughout the period but, owing to variation between cats, were significantly lower on only a few occasions. By contrast, the responses of cells to pokeweed mitogen (PWM) were severely affected and declined progressively throughout the 4-year period. In general, responses to Con A but not PWM could be restored by the addition of exogenous IL-2. The decline in immune responsiveness was concurrent with a decline in feline (f)CD4+ cells and an inversion in the CD4:CD8 ratio. Peak production of IL-1, IL-6 and TNF coincided with periods of depressed immune responses. Additionally, immunodeficient responses and elevated levels of proinflammatory cytokines were concurrent with the presence of clinical signs. We conclude that, like human immunodeficiency virus (HIV), FIV infection results in significant perturbation of the immune response. Responses to PWM appear to correlate with disease progression which suggests that the CD3 pathway is affected in the earlier stages of the disease and that additional activation pathways such as CD2 may not be affected until the animal enters the acquired immune deficient syndrome (AIDS) stage of the disease.     

Studies on the role of interleukin-12 in acute murine toxoplasmosis.

Interleukin-12 (IL-12) is important in the regulation of resistance to Toxoplasma gondii in mice with severe combined immunodeficiency (SCID). The protective ability of IL-12 in SCID mice appears to be through its activity on natural killer (NK) cells to induce production of interferon-gamma (IFN-gamma). In this study we assessed the role of IL-12 in the acute stage of toxoplasmosis in immunocompetent mice. Administration of IL-12 to BALB/c mice infected with the virulent C56 strain of T. gondii remarkably delayed time to death. The protective activity of IL-12 was abrogated by administration of monoclonal antibodies to IFN-gamma or tumour necrosis factor-alpha (TNF-alpha), and by depletion of NK cells using an antisera against asialoGM1. Whereas BALB/c mice infected with the ME49 strain of T. gondii survived infection, administration of anti-IL-12 to infected mice resulted in 100% mortality accompanied by decreased serum levels of IFN-gamma. Furthermore, this treatment significantly reversed the suppression of spleen cell proliferation to concanavalin A (Con A), which is associated with the acute stage of infection, and resulted in decreased ex vivo production of IFN-gamma, IL-2, IL-4 and IL-10 in response to Con A. Our results indicate an important role for IL-12 in mediating resistance to T. gondii during acute infection in immunocompetent mice, that NK cells are required for this protective activity, and that IL-12 is involved in the immunosuppression which accompanies this infection.     

Neutralization of IL-12 demonstrates the existence of discrete organ-specific phases in the control of Leishmania donovani

IL-12 plays a key role in stimulating both innate and antigen-specific immune responses against a number of intracellular pathogens. A neutralizing anti-IL-12 monoclonal antibody (mAb) was used to define and compare the role of endogenous IL-12 in the liver and spleen of mice infected with Leishmania donovani. IL-12 neutralization both early and late in infection caused delayed resolution of parasite load, a transient decrease in IFN-γ, IL-4, TNF-α and inducible nitric oxide synthase (NOS-2) production, and suppressed tissue granuloma formation in the liver of genetically susceptible BALB/c mice. In contrast to the liver of BALB/c mice, neutralization of IL-12 had no effect on parasite burden in the spleen over the first 28 days of infection. However, IL-12 appeared to be critical for the development of mechanisms which subsequently contain the growth of persistent parasites in this organ in that neutralization of IL-12 dramatically enhanced parasite growth after day 28 of infection. Following IL-12 neutralization, the later unchecked growth of parasites in the spleen was coincident with an extensive breakdown of the tissue microarchitecture. Immunohistochemical studies revealed that IL-12 was largely produced by uninfected cells in L. donovani-infected BALB/c mice. In contrast, the course of infection in the liver and spleen of genetically resistant CBA/n mice was unaffected by the administration of anti-IL-12 mAb. These results suggest that the liver and spleen in susceptible BALB/c mice have different temporal requirements for IL-12 in controlling L. donovani infection, whereas IL-12 plays little role in either organ in resistant CBA/n mice. In addition, IL-12 appears to be involved in the generation of both Th1 and Th2 responses during L. donovani infection in BALB/c mice.     

Immunomodulation by cells of mononuclear phagocyte lineage in acute cold-stressed or cold-acclimatized mice.

Immunoregulatory states in acute cold-stressed or cold-acclimatized mice were investigated. When male C57BL/6 mice were exposed to environmental temperature of 5 degrees for 24 hr (acute cold stress), the spleen cells showed depressed proliferative responses to stimulation with concanavalin A (Con A) or lipopolysaccharide (LPS) compared with control mice (reared at 25 degrees). The proportion of Thy-1.2+ cells increased significantly in spleens from these acute cold-stressed mice. The Con A responses of T-enriched cells from acute cold-stressed mice were restored to the normal level by adding plastic-adherent cells from control mice. Further, adherent cells from acute cold-stressed mice markedly suppressed the Con A responses of control spleen cells. Thus, the plastic-adherent cells appeared to be responsible for the suppressed Con A responses. On the other hand, proliferative responses to Con A or LPS were elevated in spleen cells from mice exposed to 5 degrees for 3 weeks (cold acclimatization). A significant decrease of Thy-1.2+ cells was detected in these spleens. It was shown that the enhanced proliferative responses were attributable to functional alterations of the plastic adherent cell population but not to those of lymphoid cell population. These findings indicate that the low or high responsiveness of spleen cells to Con A observed in cold-stressed or cold-acclimatized mice, respectively, may be due to a mechanism including the contrary modulations of functions of cells of mononuclear phagocyte lineage.     

In vivo effect of isoprinosine on interleukin-2 production, lymphocyte mitogenesis and NK activity in normal and cyclophosphamide immunosuppressed mice

The in vivo effect of isoprinosine on IL-2 production, mitogen-induced proliferation and NK activity of lymphocytes from normal as well as cyclophosphamide (CY) treated mice has been investigated. Isoprinosine was given in a single dose (50 mg/kg or 5 mg/kg) to normal or CY treated mice (250 mg/kg i.p. simultaneously to isoprinosine). An enhancement of T lymphocyte proliferation and IL-2 production was observed in both cases. There was no correlation between the small effect observed in T lymphocyte proliferation and the enhancement of IL-2 levels found in the supernatants of Con A activated spleen cells, specially in normal mice. Administration of isoprinosine every day (50 mg/kg) augmented Con A induced mitogenesis, IL-2 production and NK activity in animals treated with cyclophosphamide, but not in normal mice. Isoprinosine could be of interest in a combined treatment with immunosuppressants for the restoration of certain immune functions. The effect of isoprinosine on immune responses may be mediated in part by changes in IL-2 activity.     

Low responsiveness of hepatitis B virus-transgenic mice in antibody response to T-cell-dependent antigen: defect in antigen-presenting activity of dendritic cells.

The experiments presented here were performed to evaluate immune responsiveness of hepatitis B virus (HBV)-transgenic mice (transgenic mice), as a model of HBV-carrier state to a T-cell-dependent antigen, keyhole limpet haemocyanin (KLH). The transgenic mice which were completely unresponsive to hepatitis B surface antigen (HBsAg), responded poorly to KLH. The levels of anti-KLH antibodies (Ab) produced in vivo were significantly lower in transgenic mice compared with the normal control mice at respective immunizing doses of KLH. In addition, a little or no anti-KLH Ab production was detected in culture supernatants of KLH-primed transgenic mice spleen cells. KLH-primed T cells from normal and transgenic mice induced anti-KLH Ab production from transgenic B cells in the presence of antigen-presenting spleen adherent cells (SAC) from normal mice, but not those from transgenic mice. Depletion of dendritic cells from normal mice-derived SAC completely abrogated the anti-KLH Ab response in transgenic spleen cell culture and their addition to the culture restored the response. Low efficiency of transgenic dendritic cells was demonstrated in sodium periodate (NaIO4)-induced non-specific and allogenic antigen-induced T-cell proliferation. Finally, cytofluorometric analyses showed a reduced Ia antigen expression on transgenic dendritic cells. These results indicate that low responsiveness of transgenic mice in specific-antibody response is not due to functional defects in T cells or B cells but rather to a defect of antigen-presenting activity of dendritic cells.     

Suppression of interleukin-2 production by macrophages in genetically susceptible mice infected with Leishmania major.

Spleen cells from BALB/c mice infected with 2 X 10(7) L. major promastigotes and developing progressive disease produced significantly lower levels of interleukin-2 (IL-2) in response to concanavalin A stimulation than did spleen cells from uninfected mice. In contrast, spleen cells from sublethally irradiated and infected mice, which were able to contain lesion development, produced significantly higher levels of IL-2. The increase in IL-2 production closely paralleled lesion regression. Mice protectively immunized by four intravenous injections with lethally irradiated promastigotes also produced enhanced levels of IL-2, which were sustained after challenge infection. In contrast, spleen cells from BALB/c mice given four s.c. injections of irradiated promastigotes produced high levels of IL-2 before but not after infection. These mice eventually produced levels of IL-2 indistinguishable from those of unimmunized mice with progressive disease. There is thus an inverse relation between disease progression and the ability of spleen cells to produce IL-2. Spleen cells from mice with uncontrolled disease not only produced lower levels of IL-2 but also impaired IL-2 production by normal spleen cells. The ability to inhibit IL-2 was abrogated by passing the cells through a Sephadex G-10 column, removal of plastic adherent cells, and removal of carbonyl iron-ingesting cells. Furthermore, Sephadex G-10 column-treated and plastic adherent, nonspecific esterase-positive spleen cells from mice with progressive disease were able to suppress IL-2 production by normal splenic T cells. The suppressive activity of the adherent cells was not affected by treatment with anti-Thy-1.2 antibody and complement. In contrast, adherent spleen cells from uninfected mice were devoid of such suppressor activity. The depressed IL-2 production by spleen cells from progressively infected mice could be restored to that of normal spleen cells by the addition of indomethacin to the culture. There was however, no correlation between IL-2 production and IL-1 activity in infected or immunized BALB/c mice. Thus, it appears that the suppression of IL-2 production is mediated by prostaglandins elaborated by macrophages from chronically infected mice.     

Lymphoproliferation Induced in Mouse Spleen Cells by Mycoplasma arthritidis Mitogen

Spleen cells of various mouse strains (e.g. BALB/c, C311, and CBA) reacted towards MAS (a mitogen derived from supernatants of cultured Mycoplasma arthritidis) with a marked lymphoproliferative response. This reactivity was T-cell-dependent. It was reduced by 90% after removal of macrophages by passage of the spleen cells through Sephadex G-10 columns. Addition of 2-mercaptoethanol (2-ME) to macrophage-depleted CBA spleen cells completely restored the response to MAS. Spleen cells of C57BL/6 and C57BL/10 mice were unreactive to MAS, even in the presence of macrophages, and this non-reactivity was controlled by the I-region of H-2. Other mouse strains that, similarly to C57BL/6, lack the expression of I-E on the cell surface (that is, mice of the haplotype H-2f, H-2q, and H-2s) were also non-responsive to MAS. However, the addition of 2-ME to spleen cells of non-responder mice resulted in high lymphoproliferative responses to MAS, which were as high as those of CBA spleen cells. The reaction of C57BL/6 spleen cells to MAS in the presence of 2-ME again was T-cell-dependent, as shown by data with spleen cells of homozygous nude mice and spleen cells treated by anti-thy-1 and C. A macrophage dependency of this response was also evident. When C57BL/6 spleen cells were vigorously freed of accessory cells by the use of nylon wool columns, the MAS response could no longer be restored by 2-ME.     

The immunosuppressive compound 2-acetyl-4-tetrahydroxybutyl imidazole inhibits the allogeneic mixed lymphocyte reaction by sequestration of a recirculating subpopulation of T cells.

2-acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)imidazole (THI) is an immunosuppressive component of caramel food colouring that causes lymphopenia in mice and rats by an unknown mechanism. In this study we investigated some of the affects of THI on the murine immune system. Initially we showed that splenic T lymphocytes from mice treated with 50 mg/l THI in their drinking water were unable to launch a mixed lymphocyte reaction (MLR) against allogeneic stimulator cells, and had decreased and delayed interleukin-2 (IL-2) production. However, these T cells exhibited a normal proliferative response to concanavalin A (Con A), immobilized anti-CD3 monoclonal antibody (mAb) and anti-CD3 plus anti-CD28 mAb. Furthermore, the MLR response could be restored by the addition of IL-2 to the MLR culture. Homing studies using intravenous injection of fluorescence-labelled splenocytes showed that THI treatment decreased absolute numbers of labelled T and B lymphocytes in the blood and the spleen. Furthermore, these labelled cells reappeared in the blood and the spleen when mice were taken off THI, indicating that lymphocyte recirculation and splenic homing were modified reversibly by THI treatment. Cessation of THI treatment also resulted in a rapid reappearance of MLR responsiveness in the spleen, indicating that THI treatment does not functionally impair recirculating T cells. Collectively these data are compatible with the concept that a rapidly recirculating population of T cells, which produce IL-2 in an allogeneic MLR, are lost from the blood and spleen following THI treatment, and are sequestered in other, yet to be identified, tissues.