Shielded Bone Marrow Research Articles

MIIST305 mitigates gastrointestinal acute radiation syndrome injury and ameliorates radiation-induced gut microbiome dysbiosis.

High-dose radiation exposure results in gastrointestinal (GI) acute radiation syndrome identified by the destruction of mucosal layer, intestinal epithelial barrier dysfunction, and aberrant inflammatory responses. In addition, radiation causes gut microbiome dysbiosis characterized by diminished microbial diversity, reduction in the abundance of beneficial commensal bacteria, and the spread of bacterial pathogens that trigger the recruitment of immune cells and the production of pro-inflammatory factors that lead to further GI tissue damage. Currently, there are no FDA-approved countermeasures that can treat radiation-induced GI injury. To meet this critical need, Synedgen Inc., has developed a glycopolymer radiomitigator (MIIST305) that is specifically targeted to the GI tract which acts by intercalating into the mucus layer and the glycocalyx of intestinal epithelial cells that could potentially ameliorate the deleterious effects of radiation. Male C57BL/6J adult mice were exposed to 13 Gy total body X-irradiation with 5% bone marrow shielding and MIIST305 was administered on days 1, 3, and 5 post-irradiation. Approximately 85% of the animals survived the irradiation exposure and were apparently healthy until the end of the 30-day study period. In contrast, no control, vehicle-treated animals survived past day 10 at this radiation dose. We show that MIIST305 improved intestinal epithelial barrier function and suppressed systemic inflammatory response mediated by radiation-induced pro-inflammatory cytokines. Taxonomic profiling and community structure of the fecal and colonic mucosa microbiota demonstrated that MIIST305 treatment increased microbial diversity and restored abundance of beneficial commensal bacteria, including Lactobacillus and Bifidobacterium genera, while suppressing potentially pathogenic bacteria compared with vehicle-treated animals. In summary, MIIST305 is a novel GI-targeted therapeutic that greatly enhances survival in mice exposed to lethal radiation and protects the GI tract from injury by restoring a balanced gut microbiota and effectively reducing proinflammatory responses. Further development of this drug as an FDA-approved medical countermeasure will be of critical importance in the event of a radiation public health emergency.

Delayed effects of radiation exposure in a C57L/J mouse model of partial body irradiation with ~2.5% bone marrow shielding.

Mouse models of radiation injury are critical to the development of medical countermeasures (MCMs) against radiation. Now that MCMs against hematopoietic acute radiation syndrome (H-ARS) have achieved regulatory approval, attention is shifting to develop MCMs against the adverse effects of gastrointestinal acute radiation syndrome (GI-ARS) and delayed effects of acute radiation exposure (DEARE). The C57L/J mouse model of partial body irradiation (PBI) with 2.5% bone marrow shielding (BM2.5) is being leveraged to examine both GI-ARS and DEARE effects. Within days of PBI, mice may develop H- and GI-ARS followed several months later by DEARE as a multi-organ injury, which typically involves the lung and kidney (L- and K-DEARE, respectively). The objective of this manuscript is to describe the dose response relationship and progression of radiation injury in the C57L/J mouse and to evaluate its suitability for use in DEARE MCM testing. In two separate studies conducted over 2 years, male and female C57L/J mice were exposed to PBI BM2.5 with one hindlimb shielded from radiation, representing ~2.5% bone marrow shielding/sparing. Mice were X-ray irradiated at doses ranging from 9 to 13 Gy at 10 to 12 weeks of age for the purposes of assessing ARS survival at 30 days and DEARE survival at 182 days post-irradiation. Clinical indicators of ARS and DEARE were determined by clinical observations, body weights, hematology, clinical chemistry, magnetic resonance imaging (MRI) of lung, and histopathology of selected tissues. C57L/J mice developed canonical ARS responses of hematopoietic atrophy and gastrointestinal injury resulting in dose dependent mortality at doses ≥11 Gy between 1- and 15-days post-irradiation. In animals that survived ARS, DEARE associated mortality occurred in dose dependent fashion at ≥9 Gy for both sexes between 60- and 159-days post-irradiation with histopathology examinations indicating lung injury as the primary cause of death in moribund animals. The PBI BM2.5 C57L/J mouse model reliably produced known H- and GI-ARS effects at doses greater than those resulting in DEARE effects. Because of this, the C57L/J mouse can be used to test MCMs against L-DEARE injury, while avoiding ARS associated mortality.

Sex as a Factor in Murine Radiation Research: Implications for Countermeasure Development

There is an increased threat of exposure to ionizing radiation; in the event of such exposure, the availability of medical countermeasures will be vital to ensure the protection of the population. Effective countermeasures should be efficacious across a varied population and most importantly amongst both males and females. Radiation research must be conducted in animal models which act as a surrogate for the human response. Here, we identify differences in survival in male and female C57BL/6 in both a total body irradiation (TBI) model using the Armed Forces Radiobiology Research Institute (AFRRI) 60Co source and a partial body irradiation (PBI) model using the AFRRI Linear Accelerator (LINAC) with 4 MV photons and 2.5% bone marrow shielding. In both models, we observed a higher degree of radioresistance in female animals and a corresponding radiosensitivity in males. One striking difference in male and female rodents is body size/weight and we investigated the role of pre-irradiation body weight on survivability for animals irradiated at the same dose of irradiation (8 Gy TBI, 14 Gy PBI). We found that weight does not influence survival in the TBI model and that heavier males but lighter females have increased survival in the PBI model. This incongruence in survival amongst the sexes should be taken into consideration in the course of developing radiation countermeasures for response to a mass casualty incident.

The progression of radiation injury in a Wistar rat model of partial body irradiation with ∼5% bone marrow shielding

Purpose To describe the dose response relationship and natural history of radiation injury in the Wistar rat and its suitability for use in medical countermeasures (MCM) testing. Materials & Methods In two separate studies, male and female rats were exposed to partial body irradiation (PBI) with 5% bone marrow sparing. Animals were X-ray irradiated from 7 to 12 Gy at 7–10 weeks of age. Acute radiation syndrome (ARS) survival at 30 days and delayed effects of acute radiation exposure (DEARE) survival at 182 days were assessed. Radiation effects were determined by clinical observations, body weights, hematology, clinical chemistry, magnetic resonance imaging of lung, whole-body plethysmography, and histopathology. Results Rats developed canonical ARS responses of hematopoietic atrophy and gastrointestinal injury resulting in mortality at doses ≥8Gy in males and ≥8.5 Gy in females. DEARE mortality occurred at doses ≥8Gy for both sexes. Findings indicate lung, kidney, and/or liver injury, and persistent hematological dysregulation, revealing multi-organ injury as a DEARE. Conclusion The Wistar rat PBI model is suitable for testing MCMs against hematopoietic and gastrointestinal ARS. DEARE multi-organ injury occurred in both sexes irradiated with 8–9Gy, also suggesting suitability for polypharmacy studies addressing the combination of ARS and DEARE injury.

Opaganib Protects against Radiation Toxicity: Implications for Homeland Security and Antitumor Radiotherapy.

Exposure to ionizing radiation (IR) is a lingering threat from accidental or terroristic nuclear events, but is also widely used in cancer therapy. In both cases, host inflammatory responses to IR damage normal tissue causing morbidity and possibly mortality to the victim/patient. Opaganib, a first-in-class inhibitor of sphingolipid metabolism, has broad anti-inflammatory and anticancer activity. Opaganib elevates ceramide and reduces sphingosine 1-phosphate (S1P) in cells, conditions that increase the antitumor efficacy of radiation while concomitantly suppressing inflammatory damage to normal tissue. Therefore, opaganib may suppress toxicity from unintended IR exposure and improve patient response to chemoradiation. To test these hypotheses, we first examined the effects of opaganib on the toxicity and antitumor activity of radiation in mice exposed to total body irradiation (TBI) or IR with partial bone marrow shielding. Oral treatment with opaganib 2 h before TBI shifted the LD75 from 9.5 Gy to 11.5 Gy, and provided substantial protection against gastrointestinal damage associated with suppression of radiation-induced elevations of S1P and TNFα in the small intestines. In the partially shielded model, opaganib provided dose-dependent survival advantages when administered 4 h before or 24 h after radiation exposure, and was particularly effective when given both prior to and following radiation. Relevant to cancer radiotherapy, opaganib decreased the sensitivity of IEC6 (non-transformed mouse intestinal epithelial) cells to radiation, while sensitizing PAN02 cells to in vitro radiation. Next, the in vivo effects of opaganib in combination with radiation were examined in a syngeneic tumor model consisting of C57BL/6 mice bearing xenografts of PAN02 pancreatic cancer cells and a cross-species xenograft model consisting of nude mice bearing xenografts of human FaDu cells. Mice were treated with opaganib and/or IR (plus cisplatin in the case of FaDu tumors). In both tumor models, the optimal suppression of tumor growth was attained by the combination of opaganib with IR (± cisplatin). Overall, opaganib substantially protects normal tissue from radiation damage that may occur through unintended exposure or cancer radiotherapy.

Investigating the Abscopal Effects of Radioablation on Shielded Bone Marrow in Rodent Models Using Multimodality Imaging.

The abscopal effect is the response to radiation at sites that are distant from the irradiated site of an organism, and it is thought to play a role in bone marrow (BM) recovery by initiating responses in the unirradiated bone marrow. Understanding the mechanism of this effect has applications in treating BM failure (BMF) and BM transplantation (BMT), and improving survival of nuclear disaster victims. Here, we investigated the use of multimodality imaging as a translational tool to longitudinally assess bone marrow recovery. We used positron emission tomography/computed tomography (PET/CT), magnetic resonance imaging (MRI) and optical imaging to quantify bone marrow activity, vascular response and marrow repopulation in fully and partially irradiated rodent models. We further measured the effects of radiation on serum cytokine levels, hematopoietic cell counts and histology. PET/CT imaging revealed a radiation-induced increase in proliferation in the shielded bone marrow (SBM) compared to exposed bone marrow (EBM) and sham controls. T2-weighted MRI showed radiation-induced hemorrhaging in the EBM and unirradiated SBM. In the EBM and SBM groups, we found alterations in serum cytokine and hormone levels and in hematopoietic cell population proportions, and histological evidence of osteoblast activation at the bone marrow interface. Importantly, we generated a BMT mouse model using fluorescent-labeled bone marrow donor cells and performed fluorescent imaging to reveal the migration of bone marrow cells from shielded to radioablated sites. Our study validates the use of multimodality imaging to monitor bone marrow recovery and provides evidence for the abscopal response in promoting bone marrow recovery after irradiation.

Development of A Novel Murine Model of Combined Radiation and Peripheral Tissue Trauma Injuries.

Detonation of a 10-kiloton nuclear bomb in an urban setting would result in >1 million casualties, the majority of which would present with combined injuries. Combined injuries, such as peripheral tissue trauma and radiation exposure, trigger inflammatory events that lead to multiple organ dysfunction (MOD) and death, with gastrointestinal (GI) and pulmonary involvement playing crucial roles. The objective of this study was to develop an animal model of combined injuries, peripheral tissue trauma (TBX animal model) combined with total body irradiation with 5% bone marrow shielding (TBI/BM5) to investigate if peripheral tissue trauma contributes to reduced survival. Male C57BL/6J mice were exposed to TBX10%, irradiation (TBI/BM5), or combined injuries (TBX10% + TBI/BM5). Experiments were conducted to evaluate mortality at day 7 after TBI/BM5. Serial euthanasia was performed at day 1, 3 and 6 or 7 after TBI/BM5 to evaluate the time course of pathophysiologic processes in combined injuries. Functional tests were performed to assess pulmonary function and GI motility. Postmortem samples of lungs and jejunum were collected to assess tissue damage. Results indicated higher lethality and shorter survival in the TBX10% +T BI/BM5 group than in the TBX10% or TBI/BM5 groups (day 1 vs. day 7 and 6, respectively). TBI/BM5 alone had no effects on the lungs but significantly impaired GI function at day 6. As expected, in the animals that received severe trauma (TBX10%), we observed impairment in lung function and delay in GI transit in the first 3 days, effects that decreased at later time points. Trauma combined with radiation (TBX10% + TBI/BM5) significantly augmented impairment of the lung and GI function in comparison to TBX10% and TBI/BM5 groups at 24 h. Histologic evaluation indicated that combined injuries caused greater tissue damage in the intestines in TBX10% + TBI/BM5 group when compared to other groups. We describe here the first combined tissue trauma/radiation injury model that will allow conduction of mechanistic studies to identify new therapeutic targets and serve as a platform for testing novel therapeutic interventions.

Mechanisms Involved in the Development of the Chronic Gastrointestinal Syndrome in Nonhuman Primates after Total-Body Irradiation with Bone Marrow Shielding.

In this study, nonhuman primates (NHPs) exposed to lethal doses of total body irradiation (TBI) within the gastrointestinal (GI) acute radiation syndrome range, sparing ∼5% of bone marrow (TBI-BM5), were used to evaluate the mechanisms involved in development of the chronic GI syndrome. TBI increased mucosal permeability in the jejunum (12-14 Gy) and proximal colon (13-14 Gy). TBI-BM5 also impaired mucosal barrier function at doses ranging from 10-12.5 Gy in both small intestine and colon. Timed necropsies of NHPs at 6-180 days after 10 Gy TBI-BM5 showed that changes in small intestine preceded those in the colon. Chronic GI syndrome in NHPs is characterized by continued weight loss and intermittent GI syndrome symptoms. There was a long-lasting decrease in jejunal glucose absorption coincident with reduced expression of the sodium-linked glucose transporter. The small intestine and colon showed a modest upregulation of several different pro-inflammatory mediators such as NOS-2. The persistent inflammation in the post-TBI-BM5 period was associated with a long-lasting impairment of mucosal restitution and a reduced expression of intestinal and serum levels of alkaline phosphatase (ALP). Mucosal healing in the postirradiation period is dependent on sparing of stem cell crypts and maturation of crypt cells into appropriate phenotypes. At 30 days after 10 Gy TBI-BM5, there was a significant downregulation in the gene and protein expression of the stem cell marker Lgr5 but no change in the gene expression of enterocyte or enteroendocrine lineage markers. These data indicate that even a threshold dose of 10 Gy TBI-BM5 induces a persistent impairment of both mucosal barrier function and restitution in the GI tract and that ALP may serve as a biomarker for these events. These findings have important therapeutic implications for the design of medical countermeasures.

O-037 Modulation of Inflammatory Processes in DSS-induced Colitis Models versus Gastrointestinal Acute Radiation Syndrome with AVX-470m, an Oral Polyclonal Anti-TNF Antibody

Development of improved therapies for inflammatory bowel disease (IBD) requires animal models that elucidate underlying mechanisms of disease. Acute radiation syndrome (ARS) has devastating consequences in the gastrointestinal (GI) tract driven in part by cytokine-mediated inflammation that closely resembles the pathophysiology of IBD. Tumor Necrosis Factor (TNF) is central to the dysregulated inflammatory response in IBD, and is strongly implicated in GI ARS. In this study AVX-470 m, a bovine polyclonal neutralizing antibody for murine TNF, is used to characterize the pathogenesis of GI damage in models of GI ARS and IBD. Colitis was induced in C57BL/6 mice by including 3% DSS in drinking water for 5 days. Other C57BL/6 mice were irradiated with 15.9 Gy (∼LD80/30) from a 60Co gamma source, with partial bone marrow shielding. Animals were dosed BID with AVX-470 m (10 mg/day) or saline by oral gavage for up to 14 days. In colon and jejunum tissues, relative mRNA expression levels of cytokines and immune markers was measured by qPCR (Taqman), and protein expression was analyzed by immunohistochemistry (IHC) in paraffin embedded, formalin fixed tissue sections. Both mRNA and protein levels of TNF were increased in colon and jejunum tissue, respectively, in the colitis and GI ARS models. IL-1β, IL-6, MMP9, ICAM-1, and TNFR2 mRNA levels were increased in both models, but IL12p40 was increased only in the colitis model, and no change in TNFR1 mRNA levels was detected in either model. Protein expression of MPO was increased in both the colitis and GI ARS models. These data show a remarkable correlation of inflammatory responses between these 2 models of GI injury. In contrast, CD68 and CD3 protein were increased in colon tissue in the colitis model, but neither protein was detected in jejunum in the GI ARS model, suggesting a reduced T-cell component in GI ARS, perhaps due to destruction of these cells by the high levels of ionizing radiation. In both colitis and GI ARS models, treatment with AVX-470 m resulted in a significant decrease in TNF mRNA and protein expression in colon and jejunum tissues, respectively. AVX-470 m also decreased MPO protein expression, and reduced MMP9 mRNA expression, in both models. In the colitis model, IL-6 mRNA levels were decreased by AVX-470 m treatment in colon tissue, while in the GI ARS model IL-6 mRNA levels were transiently increased in jejunum tissue. No change in mRNA levels for ICAM-1 or TNFR1 were seen in either model following AVX-470 m treatment. These studies show that inflammatory responses in colitis and GI ARS models share a common TNF-mediated neutrophilic component in the colon and jejunum, respectively, but differ with respect to immune cell involvement in response to injury. Further studies will reveal whether these differences reflect variability in timing, tissue localization, or response to the different insults. The fact that AVX-470 m is effective in both models highlights the central role of TNF in inflammation in the GI tract, and supports the therapeutic potential of an oral anti-TNF antibody as a gut-targeted treatment for multiple GI inflammatory diseases.

Assessment of Total- and Partial-Body Irradiation in a Baboon Model

This biodosimetry study used irradiated baboons to investigate the efficacy of a kinetic multiparameter (clinical, physical, and biological) approach for discriminating partial-body irradiation (PBI) and total-body irradiation (TBI). Animals were unilaterally (front) exposed to 60Co gamma rays (8 to 32 cGy min) using either TBI or vertical left hemi-body irradiation (HBI), as follows: 2.5 Gy TBI (n = 2), 5 Gy TBI (n = 2), 5 Gy HBI (n = 2), and 10 Gy HBI (n = 2). Midline tissue doses were measured at the anterior iliac crest level with an ionization chamber, and body dosimetry was performed using thermoluminescent dosimeters. Blood samples were collected before exposure and from 1 h until 200 d after irradiation. Clinical status, complete blood cell count, biochemical parameters, and cytogenetic analysis were evaluated. The partial least square discriminant analysis chosen for statistical analysis showed that the four groups of irradiated baboons were clearly separated. However, the dicentric chromosome assay may not distinguish HBI from TBI in confounding situations where equivalent whole-body doses are similar and the time of exposure is sufficient for peripheral blood lymphocyte homogenization. Interestingly, as bone marrow shielding in HBI animals prevented aplasia from happening, hematologic parameters such as the platelet count and Flt-3 ligand level helped to distinguish HBI and TBI. Moreover, the ratio of neutrophil to lymphocyte counts, creatine kinase, and citrulline levels may be discriminating biomarkers of dose or injury. Both early and delayed clinical signs and bioindicators appear to be useful for assessment of heterogeneous irradiation.

Mobilization with Cyclophosphamide Shifts Hematopoiesis From the Bone Marrow to the Spleen In Mouse.

AbstractAbstract 1590 Introduction:Spleen is an active hematopoietic tissue in adult mice. Spleen niches supporting hematopoiesis were not extensively studied yet. Spleen does not contain osteoblastic niches which are considered to be essential for long-term hematopoiesis, and splenic hematopoiesis is assumed to originate mainly from progenitors with short-term repopulating ability. Normal spleen is primarily a lymphoid organ but it also contributes to myelopoiesis by containing approximately 5% of total body multipotent progenitors CFU-S. We have studied the effect of mobilization with cyclophosphamide on trafficking of hematopoietic stem/progenitor cells (HSPC) from the bone marrow to the spleen, and dependence of normal splenic hematopoiesis on continual supply of HSPC from the bone marrow. Method:Murine congenic model C57Bl/6 Ly5.1/Ly5.2, and C57Bl/6 GFP, was used in the experiments. Kinetics of HSPC in the bone marrow and the spleen after a sublethal dose of cyclophosphamide (135 mg/kg) was measured by clonal assays (CFU-GM and CFU-S – progenitors) and by competitive repopulation assay (short- and long-term repopulating cells, STRC and LTRC). Parabiotic pairs were established from control and cyclophosphamide-treated partners in order to demonstrate HSPC exchange between hematopoietic organs. Selective irradiation of either spleen with bone marrow shielded, or irradiation of bone marrow with spleen shielded, was used to measure significance of HSPC migration from the spleen to the bone marrow during hematopoietic regeneration. Selected cytokine expression in the bone marrow and in the spleen was determined by real-time PCR. Result:Bone marrow HSPC rapidly but transiently regenerated from cyclophosphamide damage. Secondary decrease in HSPC between 5 and 7 days after cyclophosphamide in the bone marrow was accompanied with significant reduction of SDF-1 mRNA expression, mobilization of HSPC into peripheral blood and their rapid accumulation in the spleen. Spleen thus transiently became the main hematopoietic organ in the mouse by containing 69% of total body CFU-S, and also contained significantly more LTRC compared to the femur (Fig). [Display omitted] Surprisingly, there was no increase in SDF-1 mRNA expression during homing of HSPC in the spleen. Spleens of parabiotic partners were rapidly colonized by partner cells including HSPC within two weeks. Bone marrow also contained small but significant amount of partner HSPC one and 2 weeks after parabiosis establishment. In pairs made from control and cyclophosphamide treated mice, mobilized HSPC preferentially engrafted in both own and partner spleens. Spleen cellularity and progenitor content was not affected by selective spleen irradiation and was restored from shielded bone marrow 6 days after irradiation. Expansion of extramedullary splenic hematopoiesis after cyclophosphamide occurred even when HSPC were destroyed in the spleen 1 day after cyclophosphamide, i.e. the spleen expansion was dependent on the bone marrow. Irradiation of bone marrow with spleen shielded led to abruption of splenic hematopoiesis in both control and cyclophosphamide treated mice. Conclusion:There is a quantitatively significant trafficking of HSPC by means of the circulation in mice. The splenic hematopoiesis appears to be almost fully dependent on a continuous supply of HSPC from the bone marrow. This trafficking of HSPC can be enhanced by hematopoietic cell mobilization. In mice, damage caused by cyclophosphamide results in a transient, however quantitatively remarkable relocation of HSPC, including LTRC, from the bone marrow to the spleen. This accumulation of HSPC in spleen is not accompanied with increased expression of SDF-1 mRNA. HSPC circulating in peripheral blood also significantly colonize the bone marrow and actively participate in its hematopoiesis. Support: Projects LC06044 and MSM 0021620806 of the Ministry of Education, Youth and Sports of the Czech Republic. Disclosures:No relevant conflicts of interest to declare.

Multichromatic phenotypic analysis of peripheral lymphocytes in non-human primates (NHP) following total body irradiation (TBI) with 5% bone marrow shielding (BM5) (132.19)

Abstract High dose TBI of NHP leads to severe myelosuppression, neutropenia, thrombocytopenia, and immunosuppression, concurrent with an acute gastrointestinal syndrome. BM shielding mitigates these lethal effects by preserving a reservoir of unexposed hematopoietic progenitors. Rhesus macaques were exposed to either 10Gy (sub-lethal) or 11Gy (LD15 8-10 days) in the TBI/BM5 protocol and administered supportive care (fluids, antibiotics, transfusions). Peripheral blood was drawn over 150 days post irradiation (DPI) and lymphocyte subsets were assessed by flow cytometry. 10/11Gy TBI/BM5 caused a significant, early decrease in CD3+ T cells, sustained through 42 DPI, which reached baseline at 56 DPI. CD8+ T cells dominated T cell repopulation while CD4+ T cells showed a slower recovery. Most of the CD8+ T cells were CD62L+CD45RA+. CD20+ B cells reached 168% of baseline at 56 DPI and remained above baseline through 150 DPI. CD20+CD27+ memory B cell recovery kinetics were similar to B cells up to 77 DPI, with a sharp decrease at 90 DPI. CD3+ integrin α4β7+ (mucosal marker) cells recovered to 249% of baseline levels by 35 DPI and peaked at 400% of baseline at 120 DPI. Conversely, α4β7+ B cell recovery was slower, requiring 120 DPI to reach pre-irradiation levels. Functional assays will help understand the recovery pattern of lymphocyte subsets.

Permanent loss in stem cell self renewal capacity following stress to the marrow

A technique of irradiating the entire mouse except for one hind limb was developed to provide repeated proliferative demand on the stem cell pool. Animals received 200 cGY weekly for a total dose of 3,400 to 4,000 cGy. During irradiation, shielded bone marrow cellularity was similar to that of unirradiated controls. Shielded marrow colony- forming unit (CFUs) content increased while marrow CFUs self renewal capacity decreased as compared with unirradiated age-matched controls. Following irradiation experimental animals were monitored monthly for 10 to 12 months for marrow cellularity, CFUs content, and self renewal capacity. Shielded marrow cellularity and CFUs content remained elevated over age-matched controls throughout the period of observation. These findings are compatible with the requirement of the shielded hind limb to provide hematopoietic support for the remainder of the animal. Shielded marrow self renewal capacity, a measurement reflecting primitive hematopoietic stem cell function, remained depressed and did not recover with time. These experiments provide evidence for there being limitations on the self renewal capacity of the stem cell compartment. While the small amount of shielded marrow had sufficient capacity to support the animal its average self renewal capacity was permanently reduced.

Lectin separation of nonlymphoid suppressor cells induced by total lymphoid irradiation.

Suppression of the mixed lymphocyte reaction (MLR) exerted by splenocytes derived from mice treated with fractionated total lymphoid irradiation (TLI, 200 rds x 8) was analyzed by various criteria in order to characterize the phenotype of the cell type(s) responsible for suppression. TLI-induced suppressor cells could not be eliminated by removal of cells bearing surface immunoglobulin, Thy-1, Lyt-2 and TL, and thus could not be ascribed to lymphocytes of the B or T cell lineage. Suppressor cells were large, and nonadherent to nylon wool, Sephadex G-10 and plastic surfaces. Suppressor activity of TLI splenocytes was predominantly located in fractions of cells bearing receptors for soybean agglutinin (SBA), peanut agglutinin (PNA) or both lectins. SBA+, PNA+, sequentially agglutinated (SBA followed by PNA) SBA+PNA+ and (PNA followed by SBA) PNA+SBA+ suppressor cells were radioresistant upon exposure to 1000 rds in vitro. Cells bearing the receptor for PNA but lacking that for SBA (PNA+SBA-) had sharply reduced suppressor activity. However, a radiosensitive PNA- suppressor cell subset was also documented in the spleen of TLI-treated mice. Thus, suppressor cells could best be physically separated from nonsuppressors by the SBA lectin. SBA+ suppressor cells were found, by scatter analysis, to include the population of large cells characteristic of TLI splenocytes, whereas SBA- cells were much smaller and almost exclusively devoid of suppressive capacity. The PNA receptor was found to further dissect the SBA+ suppressor cells into two distinct subpopulations: radioresistant SBA+PNA+ cells and radiosensitive SBA+PNA- cells. In summary, we suggest here the presence of at least two suppressive populations induced by TLI: radioresistant SBA+, PNA+, SBA+PNA+ or PNA+SBA+ cells, and radiosensitive PNA- and SBA+PNA- cells. Similar subsets of MLR suppressor cells can be isolated from normal bone marrow cells and splenocytes of nude mice, suggesting that suppression is mediated by large, immature, nonlymphoid cells which might migrate from shielded bone marrow compartments into the spleen of TLI-treated mice.

A partial characterization of suppressor cells in the spleens of mice conditioned with fractionated total lymphoid irradiation (TLI).

Total lymphoid irradiation (TLI) is a highly effective modality for inducing immunosuppression and transplantation tolerance. The cellular basis for this immunosuppression is not clear, although T cells have been implicated. To study further the effect of TLI on the immune system, we have examined the B cells and suppressor cells in the spleens from TLI-conditioned mice. Our results indicate that after TLI, the spleen is rapidly repopulated with many large, immature cells. The probable source of these cells is the shielded bone marrow (BM). The B cells from TLI-conditioned mice are transiently immature and hyporesponsive in vitro to a T-independent antigen. Spleen cells from TLI-conditioned mice nonspecifically suppress the in vitro T-independent anti-TNP response of normal B cells. The suppressor cells lack both B and T cell markers and adhere to Sephadex G-10. The suppressor cells in spleens from TLI-treated mice bear a number of similarities to those present in normal BM. When normal BM cells were analyzed by indirect immunofluorescence for the presence of the Mac-1 antigen, two populations of suppressor cells could be identified: one was Mac-1+ and the other was Mac-1-. These data are consistent with the possibility that a subpopulation of the suppressor cells found in normal BM and in the spleens from TLI-conditioned mice are immature cells of the monocytic/granulocytic lineage.

Origin, Kinetics, and characteristics of pulmonary macrophages in the normal steady state.

Pulmonary macrophages of mice in the steady state were isolated by lavage with PBS containing EDTA and subsequent enzymatic digestion of tissue with pronase and DNA-ase. By this method, the total pulmonary macrophage population was obtained in two cell suspensions, one with a pure population of pulmonary alveolar macrophages (PAM) and the other with a mixed population of pulmonary alveolar and pulmonary tissue macrophages (PTM). The morphological, cytochemical, and functional characteristics of both PAM and PTM were like those of mature tissue macrophages except for the presence of C3 receptors. These receptors were almost absent on PAM and present on a larger number of cells in the mixed population of PAM and PTM. The total pulmonary macrophage population of mice in the steady state is approximately equal to 2 x 10(6), of which about 93% are PAM and about 7% are PTM. In labeling experiments with 3H-thymidine, the low in vitro labeling indices (less than 3%) for both PAM and the mixture of PAM and PTM, showed that both are essentially nondividing cells. In vivo labeling studies showed an increase in the number of labeled macrophages that can only be attributed to labeled monocytes migrating into the lungs. Additional evidence was provided by a decrease in the labeling indices of pulmonary macrophages when mice were treated with hydrocortisone acetate, which causes a severe monocytopenia, thus preventing monocyte influx into the lungs. Confirmation of the bone marrow origin was obtained in mice labeled after x-irradiation with partial bone marrow shielding: labeled pulmonary macrophages were found in the exposed lungs. In all experiments, the labeling indices were identical in the two macrophage populations isolated. These results show that the influx of monocytes is the source of cell renewal for the pulmonary macrophages. No indications for an interstitial division or maturation compartment in the lung were found. Quantitation of the efflux of labeled monocytes from the blood, and the number of labeled pulmonary macrophages, showed that in the steady state about 15% of the monocytes leaving the circulation become pulmonary macrophages and that the turnover time of pulmonary macrophages is approximately equal to 27 d.

Depression of T lymphocyte function in chimpanzees receiving thymectomy and irradiation.

SUMMARY In studies analogous to those in which the thymus dependency of immune functions in murine systems was determined, three chimpanzees were thymectomized, splenectomized, exposed to lethal doses of whole body X-irradiation with limited bone marrow shielding, and subsequently evaluated for lymphocyte markers and functions over a period of years. In the oldest animal studied (Irena, 7.2 years at surgery), the percentage of peripheral blood T cells decreased to about 60% of control values and remained at that level for approximately 11/2 years before returning to normal. In the two youngest chimpanzees (Adrian and Boris, 1.7 and 2.2 years at surgery), T cell rosette values dropped to 15 to 40% of control values after irradiation. T cell percentages in one of these young chimpanzees (Adrian) returned to about 75% of the controls 11/2 years after X-irradiation. Phytohemagglutinin and concanavalin A mitogen responses were less affected in the oldest chimpanzeee. However, even in the oldest animal, the responses to phytohemagglutinin and concanavalin A began to show a gradual and consistent decline 11/2 years after irradiation. Mixed leukocyte culture responsiveness was most affected by the experimental procedures, being greatly reduced in all three chimpanzees during varying time intervals. In general, the effects of the experimental procedures used to produce T cell deficiencies varied with the age of the chimpanzee at surgery, the time after irradiation when the animal was tested, and the lymphocyte marker or function studied.

Abscopal effect of irradiation on haemopoietic stem cells of shielded bone marrow--role of migration.

Immediately after partial-body irradiation, the number of colony-forming units (CFU) of the protected marrow decreases. This decrease seems to be dose-dependent and cannot be completely explained by migration for the following reasons: there is no evidence of CFU entry to irradiated territories and the number of circulating CFU does not increase. A more plausible explanation for the phenomena is an accelerated differentiation during the first 15 min after irradiation. The migration of CFU to the spleen is observed between 15 min and 3 hours after irradiation, after which it ceases. The absence of migration 3 hours after irradiation may be due to one of the two possibilities: (1) a depletion of the mobile pool of CFU if one admits that such a pool is responsible for migration in the organism; (2) a depletion of a temporarily increased stimulus due to irradiation.

B cells in the appendix and other lymphoid organs of the rabbit: Stimulation of DNA synthesis by anti-immunoglobulin

Lymphocytes from rabbit lymphoid organs were cultured in the presence of class specific anti-immunoglobulin sera and of anti-allotype sera. Stimulation of tritiated thymidine uptake into DNA was taken to indicate the presence of the corresponding immunoglobulins on the cell surfaces. Thymus and bone marrow lymphocytes were unresponsive to all reagents tested. Popliteal lymph node contained cells responsive to anti-μ, anti-γ, and anti-α and therefore presumably bearing IgM, IgG, and IgA. Spleen had only IgM- and IgG-bearing-cells, and the appendix contained cells with IgM and IgA receptors only. The lymph node, spleen, and appendix cells of rabbits depleted of B lymphocytes by irradiation (900 R) and injection of thymocytes were unresponsive to anti-immunoglobulin but were stimulated at almost normal levels by PHA and Con A. T cell-depleted animals (thymectomy, irradiation with three divided doses of 450 R and bone marrow shielding) had immunoglobulin-bearing lymphocytes but were unresponsive to the mitogens. However a moderate level of mitogen-responsiveness reappeared by 3–4 wk after irradiation. Cells of morphologically distinct regions of the appendix, separated manually, showed different responses corresponding to the inferred origins of these anatomic areas. The “dome” and “corona” contained functional IgM- and IgA-bearing cells. The “TDA” reacted well to PHA, Con A, and PWM, but was depleted of immunoglobulin-bearing cells. The “follicle” cells, which are almost all in active DNA synthesis or mitosis, were relatively unresponsive to either T or B cell stimuli. Anti-allotype serum stimulated the same populations which responded to class-specific heteroantisera but at a slightly lower level. It was inferred that gut-associated lymphoid tissues like the appendix may play a special role as an amplification site for B-cells destined to produce IgM and IgA elsewhere in the organism.

The Factors Controlling Stem Cell Recirculation. I. Migration of Hemopoietic Stem Cells in Adrenalectomized Mice

The influence of bilateral adrenalectomy on hemopoietic stem cell (CFU) migration in mice has been studied. Formation of endogenous spleen colonies in lethally irradiated, leg-shielded mice was sharply increased by prior adrenalectomy, and this increase was not dependent on the volume of shielded bone marrow. Adrenalectomy was shown to increase endogenous spleen colony formation in sublethally irradiated mice as well. However, it had no affect on formation of spleen colonies in lethally irradiated mice injected with syngeneic bone marrow. The CFU content of murine bone marrow decreased acutely after removal of the adrenals, and this decrease was accompanied by a concomitant increase in the peripheral blood and splenic CFU. Thus, adrenalectomy appeared to have no affect on the splenic plating efficiency or proliferative rate of hemopoietic stem cells, but it did result in increased migration of stem cells from the bone marrow to the blood, and thence to the spleen. It is concluded that the adrenal steroids may be of physiologic importance in the regulation of ehmopoietic stem cell migration.