Splenic Cellularity Research Articles

Evaluation of toxicity of β-tethymustine, a new anticancer compound, in mice

The toxicity of β-tethymustine, a potential anticancer compound 1 ((Cancer Lett., 119 (1997) 7–12) was assessed in normal as well as in Ehrlich ascites carcinoma (EAC), Sarcoma-180 (S-180) and Dalton's Lymphoma (DL) tumour-bearing Swiss male mice by measuring drug-induced changes in haematological parameters, femoral bone marrow cellularity and splenic cellularity on days 9, 15 and 21 following drug treatment at the optimum dose of 8.0 mg/kg body weight from days 1 to 7. Detailed studies were also made by noting sequential changes in the above parameters in normal and EAC-bearing mice on days 12 and l8, respectively. The results indicate that the compound did not adversely affect haematopoiesis as it was observed that no significant decrease in haematological parameters and femoral marrow cellularity occurred in treated groups. Initial hyposplenic activity was, however, noted in EAC and normal treated groups on day 9 which soon reached normal count within 7–10 days after termination of drug therapy. Drug-induced hepatotoxicity and nephrotoxicity were also sequentially evaluated in normal and tumour-bearing mice on days 9, 15 and 21 but no such toxicities were detected. Also, body weight, skin and hair texture, and behavioural pattern (food and water intake and activity) did not reflect any toxic reaction in host mice at this optimum dose.

The interferon-inducible chemokines MuMig and Crg-2 exhibit antiviral activity In vivo.

MuMig (murine monokine induced by gamma interferon) and Crg-2 (cytokine responsive gene 2) are two murine chemokines of the CXC family that are induced by the interferons (IFNs): MuMig specifically by IFN-gamma and Crg-2 by IFN-alpha, IFN-beta, and IFN-gamma. To investigate the biological roles of these chemokines, recombinant vaccinia viruses (rVVs) encoding either MuMig or Crg-2 were constructed. In vitro, the chemokine-encoding rVVs replicated to similar levels to the control virus. Athymic nude mice inoculated with 10(5) PFU or less of VV-HA-Mig or VV-HA-Crg-2 resolved the infection successfully whereas mice given a similar dose of the control virus VV-HA-TK died from generalized infection. At higher doses, there was mortality in all groups but death was significantly delayed in mice infected with either chemokine-encoding rVV compared with those infected with the control virus. Virus-encoded MuMig and Crg-2 enhanced the cytolytic activity of NK cells and splenic cellularity by two- to threefold and resulted in significant increases in mononuclear cell infiltration in the livers of mice. Using specific neutralizing or depleting antibodies, we have established that the control of rVV replication in athymic nude mice, as a consequence of virus-expressed MuMig and Crg-2, requires NK cells and IFN-alpha, IFN-beta, and IFN-gamma.

Host Response to Mycobacterial Infection in the Alcoholic Rat: Male and Female Dimorphism

LI, X., C. J. GROSSMAN, C. L. MENDENHALL, P. HURTUBISE, S. D. ROUSTER, G. A. ROSELLE AND P. GARTSIDE. Host response to mycobacterial infection in the alcoholic rat: Male and female dimorphism. ALCOHOL 16(3) 207–212, 1998.—Increased susceptibility to tuberculosis occurs in the alcoholic. One explanation for the altered susceptibility is a change in T-lymphocyte modulation. To evaluate this, 24 male and 24 female Sprague–Dawley rats were treated with either a Lieber-type liquid ethanol diet (LED) or an isocaloric control (LCD). After 2 weeks, half the subjects were infected with BCG (10 8 colony-forming units) and sacrificed after 42 days. Splenic helper (CD4) and suppressor/cytoxic (CD8) cells were quantitated by flow cytometry. By three-way analysis of variance, splenic cellularity was significantly increased by infection ( p < 0.0001) but suppressed by LED ( p = 0.0002). There was a marginal sexual difference ( p = 0.065) with females exhibiting a 35% lower response while on alcohol. Examining lymphocyte subsets, the most significant changes were observed after infection (BCG) and alcohol treatment (LED). CD4 levels were deminished by LED ( p = 0.0002) but markedly increased by infection ( p < 0.0001), producing a highly significant interaction that affected both absolute number ( p < 0.0001) and relative percent present ( p = 0.0078). CD8 was influenced only by infection ( p < 0.0001). This resulted in a infection-related increase in the CD4/CD8 ratio which was lower with LED ( p = 0.0032). Splenic T-lymphocytes, predominately CD4, are involved in the host response to BCG hepatitis and are adversely influenced by LED, which may contribute to increased susceptibility. Published by Elsevier Science Inc.

Long-term haematological alterations in female B6C3F1 mice treated with busulphan

Female B6C3F1 mice (12–14 weeks old) were dosed with busulphan (BU) by the intraperitoneal route at dose levels between 10 and 40 mg/kg on four occasions at 14 day intervals. Six weeks after the final dose, mice were given normal drinking water or drinking water containing chloramphenicol succinate (CAPS) at 4 mg/ml. Animals were killed at eight timepoints after the final BU dose, the last samples being taken at day 485 or 497 and a range of haematological parameters measured. During the experiment, no differences could be detected between mice receiving BU and CAPS and those receiving BU alone. Animals surviving to the end of the study displayed moderate leucopenia but no reduction in marrow cellularity nor was any effect on erythropoiesis apparent. Lymphocyte numbers were reduced in peripheral blood and bone marrow, and splenic cellularity was also reduced. Increased mortality was seen in animals which had received 40 mg BU/kg. In the first four months after BU dosing, animals killed due to ill health were pancytopenic with hypocellular marrows; deaths that occurred thereafter were due to lymphoma.

Suppression of in vitro IFN- interferon-γ, production by spleen cells of Plasmodium chabaudi-infected C57BL/10 mice exposed to dexamethasone at a low dose

After infection with Plasmodium chabaudi, C57BL/10 mice produced significant amounts of serum IFN- γ, developed a low level of parasitemia and survived the infection. Production of IFN- γ was also obvious when spleen cells of the infected mice were stimulated with the parasite antigen contained in an erythrocyte lysate in vitro. Depletion of CD4 + T cells abrogated production of IFN- γ, leading to loss of resistance to the infection. The CD4 + T cells/IFN- γ-dependent resistance of the C57BL/10 mice against P. chabaudi was applied to assess immunotoxicological effect of dexamethasone (DEX). Administration of a high dose (0.75 mg/kg) resulted in loss of the splenic cellularity, remarkable decrease in serum IFN- γ production, increased level of parasitemia, and eventual death of the infected mice. In contrast, DEX at a low dose (0.02 mg/kg) induced no alternation in the in vivo host immune activity and the mice survived the infection. However, when spleen cells were obtained from the infected mice administered the low dose of DEX and stimulated in vitro with the parasite antigen, a significantly decreased level of IFN- γ was demonstrated with compared to control mice. These findings demonstrate that the in vitro production of IFN- γ by spleen cells from P. chabaudi-resistant C57BL/10 mice was more sensitive to the immunosuppressive effect of in vivo administration of DEX. This ex vivo assay might provide a method for evaluation of drug-induced immunotoxicity at a higher sensitivity than the conventional host resistant assays such as comparison of severity of disease or time to death.

Differential effects of stress-induced adrenal function on components of the herpes simplex virus-specific memory cytotoxic T-lymphocyte response

We have previously demonstrated in a murine model system that psychological stress, applied in the form of physical restraint, suppresses both the activation of splenic-derived, herpes simplex virus (HSV)-specific memory cytotoxic T-lymphocytes (CTLm) to the lytic phenotype and the production of cytokines associated with CTL activation and function. In the studies described herein, we investigated the hypothesis that an adrenal-dependent event is responsible, either in whole or in part, for these observations. While adrenalectomy was shown to abrogate stress-induced suppression of both HSV-specific CTLm activation and the production of IL-6 and IFN- γ, the reduction in splenic cellularity associated with restraint stress remained. In addition, a role for adrenal function in the regulation of splenic cellularity and IFN- γ production in non-stressed mice was observed. Together, these results indicate that both adrenal-dependent and adrenal-independent events, operative under both baseline and stress conditions, mediate control of the memory component of the cellular immune response to HSV infection. Overall, these studies provide insight into the mechanisms by which psychological stress modulates immune responsiveness to viral pathogens.

Primitive stem cells alone mediate rapid marrow recovery and multilineage engraftment after transplantation.

The engraftment of hematopoietic stem and progenitor cells in lethally irradiated mice was evaluated following transplants of enriched hematopoietic cell populations which were defined by surface antigen and rhodamine-123 staining. Phenotypically defined long-term repopulating stem cells, short-term pluripotent progenitors, and committed myeloerythroid progenitors all rapidly reconstituted splenic cellularity and peripheral red blood cells after transplant into myeloablated animals. In contrast, marrow cellularity was reconstituted only after transplant of long-term repopulating stem cells. In addition, peripheral blood platelet and lymphocyte counts increased only after transplantation of the long-term repopulating population. Transplantation of highly enriched multipotent progenitors resulted in a transient increase in peripheral blood myeloid cells that occurred with kinetics similar to that seen after transplant of the primitive stem cell population. Erythroid reconstitution was similar in all groups, suggesting that the effect of myeloerythroid progenitor cells in mouse marrow transplants is primarily on reconstitution of the erythroid lineage due to splenic hematopoiesis. Collectively, these results suggest that the cells which function to rapidly reconstitute the nucleated blood cells in a transplant setting are more closely related to primitive, marrow-homing stem cells than to committed progenitor cells.

Tumoricidal Activity of Lymphokine-Activated Killer Cells During Acute Protein Restriction in the Cotton Rat ( Sigmodon hispidus)

Habitat-induced alterations of immune system function have been implicated in the regulation of survival rates in wild herbivore populations. Protein availability in the diet has been shown to fluctuate with density and influence immunity in hispid cotton rats ( Sigmodon hispidus), a common herbivorous rodent of the southeastern United States. In this study, we examined the impact of short-term, moderate restrictions in dietary protein on the tumoricidal activity of lymphokine-activated killer (LAK) cells in the spleen of subadult male cotton rats in captivity. Animals were fed complete, isocaloric diets containing either 20% casein (high quality diet), or one of three moderate levels of protein (10, 8, or 5% casein) for two weeks prior to assessing LAK cell activity in vitro in the presence of YAC-1 tumor cells. Moderate restrictions in protein resulted in depressed body growth, although all animals gained mass during the second week of the trial, without significant increases in food intake. Immune organ development and cellularity were suppressed in moderately restricted cotton rats when compared to those on a high quality diet. Tumoricidal activity of LAK cells against YAC-1 targets were significantly altered by diet treatments, being elevated in the group fed a diet containing 10% casein. There was a general tendency for increased LAK cell activity among those fed one of the three moderate quality diets, but observed suppressions in splenic cellularity tended to result in a slight decline in total lytic capacity of spleens.

Impairment of T and B cell development by treatment with a type I interferon.

Type I interferons alpha and beta, naturally produced regulators of cell growth and differentiation, have been shown to inhibit IL-7-induced growth and survival of B cell precursors in vitro. After confirming an inhibitory effect on B lymphopoiesis in an ex vivo assay, we treated newborn mice with an active IFN-alpha2/alpha1 hybrid molecule to assess its potential for regulating B and T cell development in vivo. Bone marrow and splenic cellularity was greatly reduced in the IFN-alpha2/alpha1-treated mice, and B lineage cells were reduced by >80%. The bone marrow progenitor population of CD43+B220+HSA- cells was unaffected, but development of the CD19+ pro-B cells and their B lineage progeny was severely impaired. Correspondingly, IL-7-responsive cells in the bone marrow were virtually eliminated by the interferon treatment. Thymus cellularity was also reduced by >80% in the treated mice. Phenotypic analysis of the residual thymocytes indicated that the inhibitory effect was exerted during the pro-T cell stage in differentiation. In IFN-alpha/beta receptor-/- mice, T and B cell development were unaffected by the IFN-alpha2/alpha1 treatment. The data suggest that type I interferons can reversibly inhibit early T and B cell development by opposing the essential IL-7 response.

Alterations in Immune Parameters Associated with Low Level Methylmercury Exposure in Mice

Methylmercury (MeHg) is a known toxicant and continues to be a significant environmental contaminant. While the neurotoxicity and developmental toxicity of MeHg are well established, the immunotoxic effects of MeHg are just now being studied and described. This study evaluated strain and gender specific effects of low level, prolonged MeHg exposure in mice. Mice were exposed to MeHg in the drinking water (0, 3 or 10 ppm) for 4 weeks. Splenocytes and thymocytes were evaluated for alterations in immunophenotype, GSH levels, and intracellular Ca2+ flux after mitogen stimulation. MeHg exposure resulted in alterations in splenocyte and thymocyte subsets and a dose dependent decrease in GSH levels (as measured by monochlorobimane fluorescence and flow cytometry) of all splenocyte subsets. This decrease in GSH was further confirmed by biochemical assay in splenocytes. In addition, there was a dose response related decrease in mitogen stimulated Ca2+ flux and in the percentages of CD4+ splenocytes and CD8+ splenocytes from mice exposed to 10 ppm of MeHg. These results suggest that low-level chronic MeHg exposure may cause immune disfunction by disturbing thiol redox balance, transmembrane signaling and splenic cellularity.

Brazilin modulates immune function mainly by augmenting T cell activity in halothane administered mice.

Previously we reported that brazilin, the main principle of Caesalpinia sappan, was able to improve the altered immune functions caused by halothane administration in mice. To elucidate the mechanisms of its immunomodulating activities, the effects of brazilin on the functions of T cells and splenic cellularity were investigated. Brazilin decreased splenic cellularity and IL-2 production which had been augmented in mice treated with halothane (21.5% in olive oil, 10 mmol/kg) for 4 consecutive days whereas the reduced expression of IL-2 receptors by ConA or standard IL-2 was increased by brazilin treatment. These data indicate that halothane induced a dysfunction of T cells resulting in abnormal immune responses and these altered immune functions might be improved mainly by affecting the function of T cells.

SUBACUTE EXPOSURE TO 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN GERIATRIC MICE: EFFECTS IN THYMIC, SPLENIC, AND BONE MARROW HEMATOPOIETIC CELLS

Exposure of geriatric m ice (21- 1 m o) to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produced alterations in thymus, spleen, and bone m arrow hematopoietic cell populations. Female C57BL/ 6 mice were dosed (via intraperitoneal injection) with 0, 5.0, or 10.0 ug/ kg TCDD in corn oil for 5 consecutive days. Evaluation of imm une endpoints took place on the second day following cessation of dosing. A limited but significant reduction in bone marrow cellularity was produced by exposure to TCDD at 10.0 ug/ kg, in the presence of unaltered percentages of cells expressing CD45, CD45R, and Mac-1 antigens. A lim ited increase in splenic cellularity was also present at the higher dose level of TCDD, where trends toward reduced percent++ ages of CD45R B cells and Thy 1.2 T cells were observed but did not reach significance. Pronounced, dose-dependent thymic hypocellularity was evident at both levels of chemical exposure in the aged m ice, and was accom panied by+ + decreased percentages of CD4 CD8 (double positive, DP) cells and concomitant--- + increased percentages of CD4 CD8 (double negative, DN) and CD4 CD8 (single positive, SP) cells. The total numbers of thymocytes in all four phenotypes defined by CD4 and CD8 antigens decreased significantly in treated animals, an apparent reflection of the chem ical-induced hypocellularity. These results indicate that, sim ilar to perinatal and young adult m odels, the geriatric murine thym us may be a highly sensitive target of TCDD exposure.

Patterns of organ-specific engraftment by stem cell subsets and committed progenitors.

The kinetics of blood and organ engraftment following transplants of defined populations of hematopoietic stem/progenitor cells were investigated utilizing cell populations defined by surface antigen and rhodamine-123 staining. While long-term repopulating stem cells, short-term multipotent progenitors and committed progenitors all reconstituted peripheral blood red cells and splenic cellularity, only the population of cells that includes highly enriched long-term repopulating stem cells (Thy-1.1lowLinnegSca-1+Rh123low) reconstituted marrow cellularity. In addition, peripheral blood platelet and nucleated cell count increased only after transplant of the long-term repopulating population. These results argue that the major cell population that functions to reconstitute hematopoiesis after bone marrow transplantation is a primitive, marrow-homing stem cell. Transplantation of highly enriched multipotent progenitors that lack long-term reconstituting potential had no impact on hematopoietic recovery, apart from a transient increase in circulating erythrocytes. These results suggest that the primary cell population that functions to reconstitute hematopoiesis in a transplant setting is the long-term repopulating stem cell. This observation is discussed in the context of the normal hematopoietic process.

Effects of Recombinant Human Insulin‐like Growth Factor‐1 and Recombinant Human Growth Hormone on Anabolism and Immunity in Calorie‐Restricted Alcoholic Rats

Patients with severe alcoholic liver injury exhibit very low serum insulin-like growth factor-1 (IGF-1) concentrations, along with many of the symptoms that might occur with an IGF-1 deficiency state (including severe protein calorie malnutrition and immunosuppression). This study was performed to assess the effects of recombinant human (rh) IGF-1 and/or rh growth hormone (rhGH) on anabolism and immunity in the calorie-restricted, immunosuppressed alcoholic rat. Undernutrition was induced by calorie restriction such that each animal consumed 40% of ad libitum-fed controls. Alcohol was administered orally in the diet such that the mean daily intake was 9.4 g/kg/day. rhIGF-1 was administered by continuous subcutaneous infusion (380 micrograms/day) using a 14-day miniosmotic pump; rhGH was given by subcutaneous injections (400 micrograms/day). Matching placebo groups were also studied. On this regimen, ad libitum-fed controls were well nourished and increased body weight 34%, whereas Restricted controls lost 7.7% and Restricted alcohol-fed rats lost 15.2%. Significant but incomplete reversal of undernutrition was achieved with hormone therapy. Best improvement was obtained with combined therapy: rhIGF-1 + rhGH (p < 0.005; placebo versus active treatments). Immunologic impairment was observed to be severe in both thymus and spleen. The most severe changes were seen in thymi of the calorie-restricted, alcohol-fed rats, wherein 98% of the T lymphocytes were lost. rhIGF-1 treatment, but not rhGH, produced significant improvements in thymus. This was most pronounced in control rats (p < 0.005). Splenic T lymphocytes were less impaired and were more responsive to rhIGF-1 treatment; there was a maximum loss of 71% of T cells in Restricted, alcohol-fed rats. rhIGF-1 treatment completely restored splenic cellularity, as well as each of the T lymphocytes studied: CD5, CD4, and CD8. Functional status of splenic T lymphocytes was assessed by blast transformation after concanavalin A stimulation. Calorie restriction did not impair significantly this function in controls [Lieber-DeCarli control diet (LCD)]. However, it was significantly impaired in the Restricted, alcohol-fed rats (p = 0.003). In the presence of continued calorie restriction and alcohol, this function was not restored with either hormone (rhIGF-1 and/or rhGH). Their role in facilitating functional recovery after calories is restored, and alcohol is discontinued is under investigation.

Interleukin-7 enhances cell-mediated immune responses in vivo in an interleukin-2-dependent manner.

We have engineered recombinant vaccinia virus vectors expressing murine interleukin-7 (IL-7) in order to study the activity of this factor during virus infection. Virus-encoded IL-7 dramatically increased splenic cellularity in infected mice and enhanced the proliferative activity of T cells and their capacity to secrete IL-2 and IL-6, but not IFN-gamma, TNF-alpha or IL-4. Numbers of splenic CD4+ and CD8+ T lymphocytes were elevated two- to threefold. IL-7 also mediated a marked enhancement of both antigen-specific and nonspecific cellular immune activity. Total splenic antiviral cytotoxic T cells (CTL), natural killer (NK), and lymphokine-activated killer cells (LAK) responses were augmented significantly in mice given VV-HA-IL-7 compared with those given control virus, with accelerated clearance of the former. The enhanced antiviral cellular immune activity mediated by IL-7 was critically dependent on IL-2 produced by the host, but occurred independently of IFN-gamma. The ability of IL-7 to induce cellular immune responses in vivo may have applications in antiviral immunotherapy, particularly in cases of immunodeficiency.

Hematopoietic augmentation by a beta-(1,4)-linked mannan.

CARN 750 (injectable acemannan) is a polydispersed beta-(1,4)-linked acetylated mannan isolated from the Aloe barbadensis plant. It has multiple therapeutic properties including activity in wound repair and as a biological agent for the treatment of neoplasia in animals as well as the ability to activate macrophages. We report herein that CARN 750 directly or indirectly has significant hematoaugmenting properties. We observed that the subcutaneous administration of CARN 750 significantly increases splenic and peripheral blood cellularity, as well as hematopoietic progenitors in the spleen and bone marrow as determined by the interleukin-3-responsive colony-forming unit culture assay and the high-proliferative-potential colony-forming-cell (HPP-CFC) assay (a measure of primitive hematopoietic precursors) in myelosuppressed (7 Gy) C57BL/6 mice. The greatest hematopoietic effect was observed following sublethal irradiation in mice receiving 1 mg CARN 750/ animal, with less activity observed at higher or lower doses. Further, CARN 750, following daily injection, has activity equal to or greater than the injection of an optimal dose of granulocyte-colony-stimulating factor (G-CSF) in myelosuppressed mice. In this comparison, significantly greater activity was observed in the splenic and peripheral blood cellularity, and in the frequency and absolute number of splenic HPP-CFC as compared to the mice receiving G-CSF at 3 micrograms/animal. CARN 750, when administered to myelosuppressed animals, decreased the frequency of lymphocytes with a concomitant significant increase in the frequency of polymorphonuclear leukocytes (PMN). However, owing to the increased cellularity, a significant increase in the absolute number of PMN, lymphocytes, monocytes and platelets was observed, suggesting activity on multiple cell lineages. The latter is the primary difference in activity as compared to G-CSF which has activity predominantly on PMN.

Interleukin-12-induced interferon-gamma inhibits hematopoiesis in vivo in mice.

In vivo administration of interleukin-12 decreases peripheral blood counts and bone marrow hematopoiesis, although in vitro IL-12 was shown to synergize with other cytokines to stimulate the proliferation and differentiation of early hematopoietic progenitors. We investigated whether the in vivo inhibition of hematopoiesis by IL-12 is indirectly mediated by IL-12-induced IFN-gamma. IL-12 was administered to wild-type or IFN-gamma receptor deficient (IFN gamma R-/-) mice. IL-12 treatment resulted in lower peripheral blood counts and a twofold decrease in bone marrow cellularity and hematopoietic progenitors in wild-type mice, but not in IFN gamma R-/- mice. Splenic weight and cellularity were dramatically increased after IL-12 administration in wild-type mice and somewhat less in IFN gamma R-/- mice. The increase was predominantly due to NK cell and macrophage infiltration in wild-type mice, and strong extramedullary hematopoiesis in IFN gamma R-/- mice. Thus, the reduction in total bone marrow cells and hematopoietic progenitor numbers following IL-12 treatment are largely indirect, mediated by IL-12-induced IFN gamma. In the absence of IFN-gamma signaling, IL-12 promotes both bone marrow and splenic hematopoiesis, consistent with its in vitro activities.

Hematologic Effects of flt3 Ligand In Vivo in Mice

We have investigated the effects of in vivo treatment with flt3 ligand (FL) on murine hematopoiesis, including mobilization of progenitors into the peripheral blood (PB). Mice were injected once daily with 10 micrograms recombinant human FL for 15 days. On days 3, 5, 8, 10, 15, and 22, mice were killed and analyzed for the number of leukocytes and colony-forming units (CFU) in bone marrow (BM), spleen, and PB. Splenic and PB cellularity increased with time in FL-treated mice. In the spleen, there was an increase in B cells, myeloid cells, and nucleated erythroid cells; in the PB, there was an increase in lymphocytes, granulocytes, and monocytic cells. The maximal number of CFU in the BM was observed after 3 days of FL treatment, giving 3.7- and 7.3-fold increases in CFU-granulocyte-macrophage (CFU-GM) and CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), respectively, compared with mouse serum albumin (MSA)-treated controls. After 8 days of FL treatment, there was a maximal 123- and 108-fold increase in splenic CFU-GM and CFU-GEMM, respectively. The maximal number CFU-GM and CFU-GEMM were seen in PB on day 10, with 537- and 585-fold increases, respectively. Burst-forming units-erythroid (BFU-E) increased in the same time frame as those of CFU-GM and CFU-GEMM in BM, spleen, and PB, although the magnitude was not as great. Primitive day-13 CFU-spleen (CFU-S) and phenotypically defined stem cells were also mobilized into the PB of FL-treated mice with similar kinetics and magnitude to that of CFU-GM and CFU-GEMM. We conclude from these studies that FL, when administered as a single agent, is a potent mobilizer of hematopoietic progenitors into the PB.

Lyphocyte migration in L-selectin-deficient mice. Altered subset migration and aging of the immune system.

Lymphocyte trafficking across high endothelial venules (HEV) of peripheral lymph nodes (PLN) is dependent upon lymphocyte expression of L-selectin. Mice that lack this adhesion molecule provide an opportunity to determine the long-term role of L-selectin-mediated migration in the maintenance of leukocyte subpopulations. HEV in L-selectin-deficient mice were phenotypically, morphologically, and functionally comparable with wild-type mice, although there was a 70 to 90% reduction in the number of lymphocytes within PLN. These lymphocytes most likely entered PLN through the afferent lymphatics, since they did not migrate into PLN of normal mice during short-term homing experiments. The impaired trafficking of lymphocytes across PLN-HEV resulted in the accumulation of memory (CD18highCD44high) lymphocytes within PLN, and also altered the distribution of lymphocyte subpopulations within other tissues. Specifically, a 30 to 55% increase in splenic cellularity occurred due to increases in both naive and memory lymphocytes. Circulating lymphocyte numbers or subpopulations were not altered in young L-selectin-deficient mice, but circulating monocyte numbers were increased nearly threefold. In contrast, older L-selectin-deficient mice had disproportionate increases of both naive and memory CD4+ T cells present within spleen and blood. These results and the finding that memory lymphocytes in wild-type mice expressed L-selectin demonstrate a requirement for L-selectin in the regulation of memory lymphocyte migration. Therefore, L-selectin-dependent pathways of lymphocyte migration are important for the normal migration of both naive and memory lymphocytes.

Phorbol ester effects on splenic lymphocyte composition and cytotoxic T cell activities of SSIN mice: a strain deficient in CD8+ T cells.

SSIN mice are considerably more sensitive to the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) in two-stage skin carcinogenesis protocols than are most other strains and stocks of mice. Experiments were performed to determine whether there was an immunological basis for this sensitivity. SSIN mice were haplotyped and found to be H-2q. T cells represented approximately 31% of the splenic cellularity of non-treated SSIN mice, but approximately 44% in BALB/c, C57BL/6, B6C3F1 and SENCAR mice. Splenic CD4+/CD8+ T cell ratios were approximately 4.2, 2.9, 2.4, 1.8 and 1.7 in SSIN, SENCAR, BALB/c, B6C3F1 and C57BL/6 mice, respectively. The unusually high ratio in SSIN spleens was the consequence of reductions in CD8+ T cells. The ratio of CD4+/CD8+ T cells in SSIN thymocytes was similiar to that measured in the spleen. The splenic cytotoxic T lymphocyte (CTL) activities of the various murine strains inversely correlated with their splenic CD4+/CD8+ ratios and their sensitivities in two-stage skin carcinogenesis protocols. Repeated in vivo topical treatment of SSIN mice with TPA caused significant decreases in splenic T cell contents, but affected neither the splenic CD4+/CD8+ T cell ratio nor the development of a CTL response upon allogeneic tumor challenge. SSIN mice also had very low splenic natural killer cell activities. Furthermore, relative to the other strains of mice, SSIN mice were poor responders upon alloantigen challenge in mixed lymphocyte response assays. These findings demonstrate that SSIN mice differ markedly from other murine stocks and strains in their splenic lymphocyte composition and in their abilities to mount some MHC-restricted and non-restricted immunosurveillance processes.