Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense.

Bacterial pneumonia is an increasing complication of HIV infection and inversely correlates with the CD4(+) lymphocyte count. Interleukin (IL)-17 is a cytokine produced principally by CD4(+) T cells, which induces granulopoiesis via granulocyte colony-stimulating factor (G-CSF) production and induces CXC chemokines. We hypothesized that IL-17 receptor (IL-17R) signaling is critical for G-CSF and CXC chemokine production and lung host defenses. To test this, we used a model of Klebsiella pneumoniae lung infection in mice genetically deficient in IL-17R or in mice overexpressing a soluble IL-17R. IL-17R-deficient mice were exquisitely sensitive to intranasal K. pneumoniae with 100% mortality after 48 h compared with only 40% mortality in controls. IL-17R knockout (KO) mice displayed a significant delay in neutrophil recruitment into the alveolar space, and had greater dissemination of K. pneumoniae compared with control mice. This defect was associated with a significant reduction in steady-state levels of G-CSF and macrophage inflammatory protein (MIP)-2 mRNA and protein in the lung in response to the K. pneumoniae challenge in IL-17R KO mice. Thus, IL-17R signaling is critical for optimal production of G-CSF and MIP-2 and local control of pulmonary K. pneumoniae infection. These data support impaired IL-17R signaling as a potential mechanism by which deficiency of CD4 lymphocytes predisposes to bacterial pneumonia.

Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice.

C57BL/6 mice genetically deficient in interleukin 15 (IL-15(-/-) mice) were generated by gene targeting. IL-15(-/-) mice displayed marked reductions in numbers of thymic and peripheral natural killer (NK) T cells, memory phenotype CD8(+) T cells, and distinct subpopulations of intestinal intraepithelial lymphocytes (IELs). The reduction but not absence of these populations in IL-15(-/-) mice likely reflects an important role for IL-15 for expansion and/or survival of these cells. IL-15(-/-) mice lacked NK cells, as assessed by both immunophenotyping and functional criteria, indicating an obligate role for IL-15 in the development and functional maturation of NK cells. Specific defects associated with IL-15 deficiency were reversed by in vivo administration of exogenous IL-15. Despite their immunological defects, IL-15(-/-) mice remained healthy when maintained under specific pathogen-free conditions. However, IL-15(-/-) mice are likely to have compromised host defense responses to various pathogens, as they were unable to mount a protective response to challenge with vaccinia virus. These data reveal critical roles for IL-15 in the development of specific lymphoid lineages. Moreover, the ability to rescue lymphoid defects in IL-15(-/-) mice by IL-15 administration represents a powerful means by which to further elucidate the biological roles of this cytokine.

RANK is essential for osteoclast and lymph node development.

The physiological role of the TNF receptor (TNFR) family member, RANK, was investigated by generating RANK-deficient mice. RANK(-/-) mice were characterized by profound osteopetrosis resulting from an apparent block in osteoclast differentiation. RANK expression was not required for the commitment, differentiation, and functional maturation of macrophages and dendritic cells from their myeloid precursors but provided a necessary and specific signal for the differentiation of myeloid-derived osteoclasts. RANK(-/-) mice also exhibited a marked deficiency of B cells in the spleen. RANK(-/-) mice retained mucosal-associated lymphoid tissues including Peyer's patches but completely lacked all other peripheral lymph nodes, highlighting an additional major role for RANK in lymph node formation. These experiments reveal that RANK provides critical signals necessary for lymph node organogenesis and osteoclast differentiation.

TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation.

The pleiotropic activities of the potent proinflammatory cytokine TNF are mediated by two structurally related, but functionally distinct, receptors, p55 and p75, that are coexpressed on most cell types. The majority of biologic responses classically attributed to TNF are mediated by p55. In contrast, p75 has been proposed to function as both a TNF antagonist by neutralizing TNF and as a TNF agonist by facilitating the interaction between TNF and p55 at the cell surface. We have examined the roles of p55 and p75 in mediating and modulating the activity of TNF in vivo by generating and examining mice genetically deficient in these receptors. Selective deficits in several host defense and inflammatory responses are observed in mice lacking p55 or both p55 and p75, but not in mice lacking p75. In these models, the activity of p55 is not impaired by the absence of p75, arguing against a physiologic role for p75 as an essential element of p55-mediated signaling. In contrast, exacerbated pulmonary inflammation and dramatically increased endotoxin induced serum TNF levels in mice lacking p75 suggest a dominant role for p75 in suppressing TNF-mediated inflammatory responses. In summary, these data help clarify the biologic roles of p55 and p75 in mediating and modulating the biologic activity of TNF and provide genetic evidence for an antagonistic role of p75 in vivo.

Flt3 ligand synergizes with granulocyte-macrophage colony-stimulating factor or granulocyte colony-stimulating factor to mobilize hematopoietic progenitor cells into the peripheral blood of mice.

Peripheral blood progenitor cells (PBPC) are increasingly being used in the clinic as a replacement for bone marrow (BM) in the transplantation setting. We investigated the capacity of several different growth factors, including human flt3 ligand (FL), alone and in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF ) or granulocyte colony-stimulating factor (G-CSF ), to mobilize colony forming cells (CFU) into the peripheral blood (PB) of mice. Mice were injected subcutaneously (SC) with growth factors daily for up to 10 days. Comparing the single agents, we found that FL alone was superior to GM-CSF or G-CSF in mobilizing CFU into the PB. FL synergized with both GM-CSF or G-CSF to mobilize more CFU, and in a shorter period of time, than did any single agent. Administration of FL plus G-CSF for 6 days resulted in a 1,423-fold and 2,717-fold increase of colony-forming unit-granulocyte-macrophage (CFU-GM) and colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) in PB, respectively, when compared with control mice. We also followed the kinetics of CFU numerical changes in the BM of mice treated with growth factors. While GM-CSF and G-CSF alone had little effect on BM CFU over time, FL alone increased CFU-GM and CFU-GEMM threefold and fivefold, respectively. Addition of GM-CSF or G-CSF to FL did not increase CFU in BM over levels seen with FL alone. However, after the initial increase in BM CFU after FL plus G-CSF treatment for 3 days, BM CFU returned to control levels after 5 days treatment, and CFU-GM were significantly reduced (65%) after 7 days treatment, when compared with control mice. Finally, we found that transplantation of FL or FL plus G-CSF-mobilized PB cells protected lethally irradiated mice and resulted in long-term multilineage hematopoietic reconstitution.

Phenotypic and functional characterization of mice that lack the type I receptor for IL-1.

IL-1 alpha and IL-1 beta bind to receptors termed the type I and type II IL-1 receptors. The type I IL-1 receptor is responsible for specific signaling, while the type II IL-1 receptor functions as a nonsignaling decoy receptor. To determine the effect of a defect in IL-1-mediated signaling, mice have been produced with a genetically disrupted type I IL-1 receptor gene. Mice lacking type I IL-1 receptors are of normal vigor and exhibit no overt phenotype. B cells from type I IL-1R-/- mice activated in vitro with anti-IgM do not proliferate in response to IL-1, but do so in response to IL-4. Injection of murine IL-1 alpha does not induce detectable serum IL-6 levels in type I IL-1R-/- mice, but equivalent levels are produced in response to LPS. Type I IL-1R-/- mice have normal serum Ig levels and generate equivalent primary and secondary Ab responses as wild-type mice. In response to LPS, acute phase protein mRNA induction are equivalent in type I IL-1R-/- and wild-type mice. Type I IL-1R-/- mice do not differ from control mice in susceptibility to either a lethal challenge with D-galactosamine plus LPS or high dose LPS. Interestingly, ICE-/-/type I IL-1R-/- double mutant mice are resistant to high dose LPS. Type I IL-1R-/- mice backcrossed to the C57BL/6 background were as equally resistant as wild-type mice to Listeria monocytogenes.

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.

Exogenous tumor necrosis factor alpha and interleukin-1 alpha increase resistance to Salmonella typhimurium: efficacy is influenced by the Ity and Lps loci.

Interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF-alpha) administered prior to infection with Salmonella typhimurium increases survival in mice that are Ityr, not in susceptible Lpsd or Itys mice. Combined IL-1 alpha and TNF-alpha pretreatment results in greater survival than that seen with either cytokine alone in Ityr mice. Treatment after infection with TNF-alpha and/or IL-1 alpha increases the mean time to death but not the survival fraction of Lpsd mice and was ineffective in either Ityr or Itys mice.

Analysis of the intra-epithelial lymphocyte compartment in SCID mice that received co-isogenic CD4+ T cells. Evidence that mature post-thymic CD4+ T cells can be induced to express CD8 alpha in vivo.

Severe combined immunodeficient (SCID) mice injected with co-isogenic CD4+/CD45RBhigh lymph node T cells from normal donors develop a wasting disease that is caused by hyperplasia of the intestinal epithelium. SCID mice injected with purified lymph node CD4+ T cells or CD4+/CD45RBlow T cells do not develop the disease. The IEL compartment from SCID mice injected with highly purified CD4+/CD45RBhigh T cells or CD4+ T cells contained significant numbers of T cells that expressed both CD4 and CD8 alpha, but not CD8 beta. The CDr+/CD8 alpha + T cells were unique to the IEL compartment of the small intestine and were not observed in significant numbers in the lamina propria, mesenteric lymph node, nor IEL compartment of the large intestine. By using Ly-5 mismatched donors and recipients, we determined that the CD4+/CD8 alpha + T cells were derived from the donor T cells. The expression of CD8 alpha was stable in vitro, and CD8 alpha mRNA was detected in sorted CD4+/CD8 alpha + T cells by reverse transcriptase-PCR (RT-PCR). Recombinase-activating gene (RAG)-1 and -2 mRNA was not detected in the intra-epithelial lymphocyte CD4+/CD8 alpha + T cell population. Thus, it appears that under conditions unique to the epithelial layer of the small intestine, mature post-thymic CD4+ T cells can be induced to express CD8 alpha.

Steel factor (c-kit ligand) stimulates the in vitro growth of immature CD3-/CD4-/CD8- thymocytes: synergy with IL-7.

The ability of Steel factor (SLF) to stimulate the growth of immature thymocytes alone and in combination with IL-7 was assessed. In suspension cultures of CD4-/CD8- thymocytes, SLF itself did not induce significant proliferation, but in combination with IL-7, it induced a greater response than did IL-7 alone. In fetal thymus lobe submersion cultures, SLF stimulated the growth of cells to a magnitude similar to that of IL-7 and the combination resulted in a synergistic response. Phenotypic analysis of these cells revealed that SLF in comparison to IL-7 stimulated the growth of a less mature population. The synergistic growth stimulated by the combination of IL-7 and SLF occurred in a population of IL-2R+/CD4-/CD8-/CD3- cells. Cells grown in SLF expressed low levels of IL-2R and overnight culture in IL-7 dramatically increased IL-2R expression. Thus, these results are consistent with the hypothesis that SLF stimulates the growth of a less mature thymocyte population than does IL-7.

Treatment of mice with IL-1 before infection increases resistance to a lethal challenge with Salmonella typhimurium. The effect correlates with the resistance allele at the Ity locus.

Administration of a single dose of IL-1 alpha to various strains of mice approximately 16 h before a lethal infection with Salmonella typhimurium resulted in a significant augmentation of survival in most Ityr, but in no Itys strains of mice. Lower numbers of bacteria were observed in the liver and spleen in response to IL-1 pretreatment shortly after infection in all Ityr strains of mice tested, including the congenic C.D2-Ityr mice. Treatment with IL-1 alpha after infection had no effect on survival in either Ityr or Itys mice. A combination of IL-1 alpha pretreatment with IL-1 alpha post-treatment did not increase survival over the effect of IL-1 alpha pretreatment alone in Ityr mice and did not increase the survival of the Itys mice. The combination of IL-1 alpha pretreatment with GM-CSF post-treatment was effective in Ityr but not in Itys strains of mice. Thus, IL-1 alpha pretreatment enhances the resistance of Ityr, but not Itys strains of mice to a lethal challenge with S. typhimurium by up-regulating antibacterial mechanisms shortly after infection.

Induction of wasting disease in SCID mice by the transfer of normal CD4+/CD45RBhi T cells and the regulation of this autoreactivity by CD4+/CD45RBlo T cells.

SCID mice injected with coisogenic CD4+/CD45RBhi lymph node T cells from normal donors develop a wasting disease that is due to hyperplasia of the intestinal epithelium. SCID mice injected with purified lymph node CD4+ T cells or CD4+/CD45RBlo T cells do not develop the disease. In addition, mixture of the CD4+/CD45RBlo T cells with equal numbers of CD4+/CD45RBhi T cells inhibits the development of disease. SCID mice that were reconstituted with CD45RBhi T cells with active disease were treated with oral antibiotics and this ameliorated the symptoms, suggesting a role of the gut bacterial flora in the development of disease. Attempts were made to accelerate or inhibit disease by chronically administering cytokines to the mice. Neither IL2 nor IL4 were effective in altering the course of disease development when given in doses known to be effective in other in vivo models. Thus, the regulation of the reactivity seen in these SCID mice may involve as yet unappreciated mechanisms.

CD4+ T cells that express high levels of CD45RB induce wasting disease when transferred into congenic severe combined immunodeficient mice. Disease development is prevented by cotransfer of purified CD4+ T cells.

Purified CD4+ lymph node T cells were sorted into two populations on the basis of their expression of CD45RB (CD45RBhi and CD45RBlo) and injected into congenic severe combined immunodeficient (SCID) mice. After a period of time that was dependent on the number of cells injected, the SCID mice that received CD45RBhi/CD4+ T cells developed a wasting disease that was not seen in SCID mice that received the CD4+/CD45RBlo cells or whole lymph node cells. At death, SCID mice that received the CD4+/CD45RBhi cells had increased spleen and lymph node cellularity compared with normal SCID mice and SCID mice that received the CD4+/CD45RBlo T cells. The spleen and lymph node contained CD4+ cells and neither CD8+ nor surface immunoglobulin M-positive cells, plus a population of cells that did not express any of those markers. At necropsy, the SCID mice that received the CD4+/CD45RBhi cells had significant hyperplasia of the intestinal mucosa with significant lymphoid cell accumulation in the lamina propria. Interestingly, mice that received mixtures of whole lymph node or purified CD4+ cells with CD4+/CD45RBhi cells did not develop weight loss, indicating that the unseparated CD4+ population contained cells that were capable of regulating the reactivity of the CD4+/CD45RBhi cells.

Interleukin-1 administration to C3H/HeJ mice after but not prior to infection increases resistance to Salmonella typhimurium.

Interleukin-1 (IL-1) treatment of C3H/HeN and C3H/HeJ mice prior to infection with Salmonella typhimurium increased the survival fraction only in C3H/HeN mice. IL-1 administration after infection resulted in a significant increase in mean survival time in C3H/HeJ but not C3H/HeN mice. Bacterial growth in IL-1-treated C3H/HeJ mice was less than that in control mice.

Administration of IL-7 to normal mice stimulates B-lymphopoiesis and peripheral lymphadenopathy.

Normal mice were injected with IL-7 (500 ng, twice daily) for various periods of time up to 6 days and the cellularity and phenotypic composition of the thymus, spleen, lymph node, and bone marrow was assessed. After 6 days of treatment, significant increases in the cellularity of the spleen, lymph node, and bone marrow were observed which returned to the normal range within 6 days after cessation of treatment. After 3 days of IL-7 treatment, increased numbers of B220+/surface(s) IgM- bone marrow cells were observed. After 6 days of treatment, these numbers were still further increased and a significant population of B220+/sIgM- cells were observed in the spleen. The numbers of c mu+/sIgM- cells were also increased in the IL-7-treated mice. Analysis of the expression of B220 and BP-1 on the sIgM- bone marrow cells revealed that the B220+/BP-1+ population was dramatically increased after IL-7 treatment and the size of the B220+/BP-1- population did not differ from control mice. The pre-B cell numbers declined rapidly after the cessation of IL-7 treatment. After 6 days of IL-7 treatment, a twofold increase in the number of B cells in the spleen and lymph node was observed. The B cell numbers declined to normal values within 6 days after the cessation of IL-7 administration. In the spleens of the IL-7-treated mice, there was a significant increase in the number of B cells with an immature phenotype (e.g., sIgMhi/sIgDlo, decreased levels of Ia and FcR expression). The numbers of CD8+ and CD4+ T cells were also increased in the lymph node and spleen of the IL-7-treated mice. These numbers declined to normal levels after the cessation of IL-7 treatment.

GM-CSF administration augments the survival of ity-resistant A/J mice, but not ity-susceptible C57BL/6 mice, to a lethal challenge with Salmonella typhimurium.

Ity resistant A/J mice were challenged with a lethal dose (2 x 10(3) organisms) of Salmonella typhimurium. Infected mice treated with 1 microgram of GM-CSF twice daily showed increased median survival time and had a higher survival fraction than untreated controls. GM-CSF was most effective when given for a brief period (1 to 2 days) after infection. Pretreatment of the mice or delayed treatment with GM-CSF had no effect on the survival of the mice. Studies on the effect of GM-CSF on the bacterial load showed that mice treated with GM-CSF had fewer S. typhimurium in the spleen and peritoneal cavity on day 4 but not on day 2 after infection. GM-CSF treatment of ity-susceptible C57BL/6 mice infected with 10 organisms had no therapeutic effect.

In vivo administration of IL-1 induces thymic hypoplasia and increased levels of serum corticosterone.

Administration of IL-1 alpha or IL-1 beta to normal mice induces a decrease in thymic cellularity, the magnitude of which depends on the number of injections and dose of IL-1. Twice daily injections of 200 ng of IL-1 alpha or -beta for 4 days results in a 90% decrease in thymic cellularity, which regenerated after cessation of treatment. Study of thymocyte subpopulations revealed that the number of CD4+/CD8+ thymocytes was dramatically decreased in IL-1-treated mice. Functional assessment of the CD4-/CD8- population from treated animals showed that these cells had adequate mitogenic responses in vitro and that the proportion of these cells in cycle was not different from control CD4-/CD8- cells. IL-1 treatment also prevented the regeneration of thymic cellularity after irradiation. The use of strains of mice differing genetically at the Ly 1 locus to construct radiation bone marrow chimeras demonstrated that bone marrow-derived thymocyte precursors were able to seed the thymus in the IL-1-treated animals. Again, however, the CD4+/CD8+ thymocyte population was significantly decreased. Thymic repopulation occurred upon cessation of IL-1 therapy. Finally, we determined that a single i.p. injection of IL-1 caused a three-fold increase in serum corticosterone levels, which peaked approximately 3 h after IL-1 administration. Thus, an IL-1-dependent increase in serum corticosterone levels may be responsible for the observed thymic hypoplasia.

The influence of IL-1 treatment on the reconstitution of the hemopoietic and immune systems after sublethal radiation.

The influence of IL-1 administration on the recovery of the hemopoietic and immune systems from sublethal irradiation was assessed. Mice were irradiated (750 R) and injected twice daily with purified recombinant derived IL-1 beta (200 ng/injection). At various times after irradiation, the functional capacity of the hemopoietic and immune systems was determined. It was found that IL-1 therapy resulted in a significantly greater number of granulocyte-macrophage-CSF responsive colony-forming cells in the bone marrow of the irradiated mice on days 5 and 11 postirradiation but not at later times. In addition the radiation induced neutropenia recovered quicker in the IL-1-treated mice with significantly greater numbers of peripheral blood granulocytes being seen on days 15 and 20 after irradiation. The influence of IL-1 therapy on the recovery of the immune system was also assessed. Of note was the observation that mice receiving IL-1 therapy had chronically hypoplastic thymi. Although thymic cellularity increased with time after irradiation in the control mice, there was no such increase in the IL-1-treated mice. Similarly, the number of pre-B cells in the marrow of these mice was also diminished. Thus, in the IL-1-treated mice the regeneration of the peripheral immune function was retarded, characterized by a general lymphopenia and decreased splenic responses to mitogenic stimuli.

Response of resident murine peritoneal macrophages to in vivo administration of granulocyte-macrophage colony-stimulating factor.

The effect of s.c. inoculation of purified recombinant derived granulocyte-macrophage (GM)-CSF on resident murine peritoneal macrophages was assessed in this study. From 18 to 24 h after s.c. administration of GM-CSF to normal mice, the resident peritoneal macrophages were harvested and the levels of membrane-bound IL-1, FcR, Mac-1 cell-surface Ag, and class II MHC expression were assessed. Peritoneal cells from GM-CSF-inoculated mice had significantly greater levels of membrane-bound IL-1 than did control mice. In addition when resident peritoneal macrophages from normal mice were purified by adherence and grown in the presence of GM-CSF, they produced greater levels of both membrane-bound and secreted IL-1. The peritoneal cells from GM-CSF-inoculated mice did not differ from controls in the expression of class II MHC-encoded Ag. This observation was confirmed by the finding that GM-CSF was unable to induce class II MHC expression on P388D1 cells, whereas a secondary mixed leukocyte culture supernatant was. Peritoneal cells from GM-CSF-inoculated mice also exhibited greater levels of expression of FcR and the Mac-1 cell-surface Ag. This resulted in an increase in their ability to phagocytose opsonized SRBC in vitro.

Cure of malignant lymphoma in dogs with peripheral blood stem cell transplantation.

Twelve dogs with spontaneous malignant lymphoma in chemotherapy-induced remission were treated with total-body irradiation (TBI) and transplantation of autologous peripheral blood mononuclear cells collected following chemotherapy-induced expansion of the stem cell pool. Four animals (33%) died of transplant-related complications, five (41%) relapsed, and three (25%) are long-term disease-free survivors. Recovery to 1000 white cells/mm3 occurred by day 16 and dogs no longer required platelet transfusions by day 37. With the exception of delayed platelet recovery these results are virtually identical to those previously reported using autologous bone marrow transplantation for canine lymphoma. This study therefore suggests that autologous peripheral blood mononuclear cells collected following chemotherapy-induced expansion of the stem cell pool may offer a realistic alternative to autologous marrow transplantation in patients with malignant lymphoma for whom autologous marrow is not available.