Prevention of death from metastases by immune RNA therapy.

The effect of immune RNA treatment on the incidence of death from pulmonary metastases was studied in C57BL/6J mice after excision of a B16 murine melanoma. Immune RNA was extracted from the lymphoid tissues of guinea pigs immunized with B16 tumor and then incubated in vitro with normal C57BL/6J mouse splenocytes. Mice receiving intraperitoneal injections of these RNA-treated syngeneic splenocytes after the primary B16 isograft was resectioned showed significantly improved long-term survival (42 to 67 percent in three successive experiments) as compared to control mice (0 to 20 percent survival) receiving untreated splenocytes. The effect of RNA treatment was tumor-specific and ribonuclease sensitive. The results suggest that immunotherapy with immune RNA may be of benefit to certain patients after surgery for cancer.

Transformation of human lymphocytes: inhibition by homologous alpha globulin.

An alpha globulin fraction prepared from normal human plasma by column chromatography prevents homologous lymphocyte transformation and the stimulation of DNA, and also protein synthesis induced by phytohemagglutinin and specific antigens. These observations support the concept of a normal circulating immunosuppressant factor which prevents lymphoid cell proliferation.

Generation of suppressor cells in mice after surgical trauma.

Immunoincompetency is often seen in patients after various types of trauma and is associated with increased morbidity and mortality from infectious complications. To understand better the immunologic impairment associated with trauma, we have studied this phenomenon in an animal model. Splenocytes from mice traumatized by amputation of their right hind limbs were consistently shown to have a diminished capacity to proliferate in response to alloantigens and to form alloreactive cytolytic cells in mixed lymphocyte cultures. Anesthesia itself had no effect in this system. The immunoincompetency was detected from 2 h to 6 d after surgical trauma and was completely reversed by removing adherent and phagocytic cells from the splenocytes. Furthermore, addition of splenocytes from traumatized mice to mixed lymphocyte cultures from normal mice prevented normal lymphocytes from responding to alloantigens, suggesting the existence of suppressor cells. The suppressor cells were found to adhere to glass and to nylon wool columns, and were contained within an esterase-positive cell population. They were insensitive to treatment with anti-Thy 1.2 and anti-Ig sera in the presence of complement. Therefore, the present results suggest that a Thy 1.2-negative, Ig-negative, esterase-positive cell population capable of adhering to glass and nylon wool, presumably macrophages, was responsible for the inhibition of the responsiveness of lymphocytes to alloantigens in traumatized animals.

Correlation of the production of plasminogen activator with tumor metastasis in B16 mouse melanoma cell lines.

The correlation between the production of plasminogen activator (PA) of tumors and their metastatic potential was studied. B16 melanoma cells and "B16 mets" cells (harvested from the pulmonary metastatic nodules of C57BL/6J mice bearing B16 isografts) were examined with respect to their fibrinolytic activity (FA) in tissue culture. B16 mets cells had a significantly higher FA than did B16 cells. F1 (a B16 subline with a lower incidence of metastasis) and F10 (a highly metastatic B16 subline) were also studied. F10 cells produced more FA than did F1 cells. The difference between the FA's of these tumors was due to differences in their PA production. Significant differences in PA production between F1 and F10 could be consistently observed when 10(5) or more cells were cultured for at least 24 hr. The cell-free supernatants harvested from 72-hr cultures of F10 cells had a higher FA than those harvested from F1 cultures. Results suggest that a quantitative difference in PA production between these 2 melanoma sublines does exist and that it may contribute to their different metastatic potential.

The specificity of concomitant tumor immunity at large tumor volumes.

Two antigenically distinct fibrosarcomas, designated BP-8 and BP-9, induced in syngeneic C3H/HeN mice by 3,4-benzo(a)pyrene were used to study tumor-specific immunity, concomitant tumor immunity and the effect of large tumor volumes on the loss of immunological reactivity. Two groups of mice were immunized to the BP-8 tumor by amputation of growing BP-8 isografts. One group was rechallenged with the BP-8 cells, and tumor growth was not noted. Both groups of mice then received an inoculum of BP-9 cells that grew to palpable tumors to the same extent as in control mice. BP-8-immunized mice bearing progressively larger PB-9 tumors were sacrified at varying intervals after the BP-9 isograft. Tumor weight was recorded as a percentage of total body weight and viable spleen cells from these animals were tested in vitro for cytotoxicity against BP-8 and BP-9 cells in the [125I]iododeoxyuridine microcytotoxicity assay. Spleen cells from untreated mice were used as controls. The mice with growing BP-9 tumors developed an immune reaction against the tumor antigens which increased with time from initial tumor isograft and with increasing tumor size up to a definite but variable limit. Cytotoxicity to BP-9 cells rose from 18% when the BP-9 tumor was not palpable to a maximum of 77% when the tumor represented 5 to 10% of the total body weight. Cytotoxicity to BP-9 fell progressively as tumor size exceeded 15% of the total body weight and approached the 10% background cytotoxicity of control lymphocytes to BP-9 cells, when the tumor weight achieved 25% of the animal's weight. Conversely, cytotoxicity of lymphocytes against the BP-8 tumor did not vary significantly and remained about 41 to 44% over the same interval even while specific reactivity to BP-9 cells significantly decreased. In addition, with time, lymphocyte-mediated cytotoxicity to the BP-8 tumor increased from 41 to 70% if the BP-8-immunized mice had been rechallenged with antigenically identical BP-8 cells prior to the BP-9 isograft. These data suggest that loss of immunoreactivity at large tumor volumes is tumor and, presumably, antigen specific. No evidence of a generalized immune paralysis was demonstrated, since the mice always maintained immunity to the BP-8 tumor despite progressive and lethal growth of the antigenically distinct BP-9 tumor.

Association of impaired immune responsiveness of lymphocytes from animals bearing large tumors with a membrane-bound suppressive substance.

The immunoreactivity of lymphocytes from mice bearing large tumors was investigated. As compared to normal lymphocytes, lymphocytes from tumor bearers manifested a lessened proliferative response to T-cell mitogens and were less capable of destroying syngeneic tumor target cells. However, the impaired reactivity of these lymphocytes was improved significantly after washing repeatedly in tissue culture medium in vitro. The increase in cytolytic activity was tumor specific. A membrane-associated suppressive substance was detected in the washing medium which inhibited the cytotoxicity of washed lymphocytes. The effect of suppressive substance was tumor specific and sensitive to treatment with protein A and anti-immunoglobulin antibody, indicating that the suppressive substance contained immunoglobulin. The present findings suggest that a membrane-associated suppressive substance(s) may be in part responsible for the diminished immune responsiveness of lymphocytes from animals bearing large tumors.

Suppression of tumor-specific cell-mediated cytotoxicity by immunoregulatory alpha-globulin and by immunoregulatory alpha-globulin-like peptides from cancer patients.

The suppression of tumor-specific cell-mediated cytotoxicity by human immunoregulatory alpha-globulin (IRA), by a peptide fraction derived from IRA, and by IRA-like peptides from the serum of cancer patients was studied in a syngeneic murine tumor-host system. Splenic lymphocytes from tumor-immunized mice were cytotoxic specific tumor cells in vitro as measured by the [125]-iododeoxyuridine release microcytotoxicity assay. However, this effect was significantly depressed if 1.25 to 5 mg of IRA per ml were added to the cultures. Pooled lyophilized normal human serum protein was inactive. IRA peptide and IRA-like peptide fractions from cancer patients were also highly suppressive of cell-mediated cytotoxicity at much lower concentrations (0.05 to 0.5 mg/ml). Control human serum peptide, which failed to inhibit the induction of hemolytic plaque-forming cells in sheep erythrocyte-injected mice, had no effect on cell-mediated cytotoxicity. IRA and IRA-like peptide fractions were not cytotoxic to the effector lymphocytes or to the target cells at the concentrations used.

Isolation of an immunosuppressive peptide fraction from the serum of cancer patients.

An immunosuppressive peptide fraction was isolated by means of gel filtration, membrane partition, and ion-exchange chromatography from the sera of patients hospitalized for cancer. The resulting peptide fraction, which was heterogeneous as judged by high-voltage electrophoresis, was found to suppress both phytohemagglutinin-induced proliferation of lymphocytes in vitro and the in vivo induction of splenic plaque-forming cells in mice. The specific activity of the peptide fraction, which was isolated from the sera of cancer patients, was significantly increased over that of the unfractionated starting material. Moreover, in control experiments, when the sera of normals or non-cancer-bearing hospitalized individuals were subjected to the same chromatographic techniques, no active peptide fraction could be obtained.

Anti-immunoglobulin as an adjunct in producing long-term antigen-specific allograft survival. Organ localization of immune complexes.

Heterologous anti-immunoglobulin is a potent immunosuppressive agent that prolongs H-2- and H-3-incompatible skin graft survival. In conjunction with antithymocyte serum, anti-immunoglobulin promotes greater graft life than either antiserum used alone, without evidence of toxicity to recipient animals. Combination treatment with anti-immunoglobulin, antithymocyte serum, and donor spleen cells produces long-term allograft survival to the lifetime of the host and can result in antigen-specific immune unresponsiveness of sufficient strength to permit acceptance of a second-set graft. Anti-immunoglobulin complexes formed in treated mice appear to be localized primarily in the lymphatic reticuloendothelial system.

The effects of injury on the adaptive immune response.

For more than thirty years it has been apparent that serious injury in humans and experimental animals is associated with a decrease in immune functions dependent upon T cells, the principal cells involved in initiating adaptive immune responses. This review focuses on more recent evidence that T helper cell function is altered after serious injury with loss of T helper 1 function and cytokine production and with preservation of T helper 2 function and an increased production of T helper 2 cytokines. Emphasis is placed on the importance of interactions between the innate and adaptive immune systems in the perturbed immune responses seen following injury. Immunomodulatory strategies are mentioned that have had success in animal models in ameliorating the diminished resistance to infection commonly seen after major traumatic or thermal injury. Finally, it is emphasized that immunomodulatory treatments that are successful in preventing infection may be contraindicated once infection is manifest.

Severe injury triggers antigen-specific T-helper cell dysfunction.

Although it is established that post-injury immune dysfunction involves alterations in T-cell function, the effects of injury on T-cell function in vivo are poorly understood. This study uses a mouse injury model and an antigen immunization approach to investigate the influence of injury on antigen-specific T-helper cell function. We report here that injury triggered a significant reduction in antigen-specific T-helper-1 (Th1)-dependent IgG2a antibody formation, while IgM, IgG1, and IgE production was unchanged. In addition, injury caused a reduction in cytokine production (IL-2, IFNgamma and IL-10) by antigen-stimulated T-cells. We also demonstrate that interleukin 12 (IL-12), a cytokine that promotes Th1 cell differentiation, restored IgG2a antibody formation and corrected the injury-induced reduction in antigen-stimulated cytokine production. Taken together, these findings indicate that severe injury induces a dramatic reduction in Th1 cell function in vivo and suggest that therapies designed to restore Th1 cell function may be beneficial to the injured host.

Injury induces alterations in T-cell NFkappaB and AP-1 activation.

The immune dysfunction that occurs after severe injury involves major changes in T-cell-mediated immunity resulting in suppressed T-helper 1 (Th1) type responses and increased or persistent T-helper 2 (Th2) type cytokine production. Since little is known about what signaling pathways are responsible for this injury-induced phenotypic shift in T-cells, we undertook this study to address the molecular basis for injury effects on T-helper cell subset cytokine expression. Experiments were designed to test whether diminished IL-2 gene expression after thermal injury coincided with changes in the induction of IL-2 gene regulatory transcription factors. Electrophoretic mobility shift assays (EMSA) were used to screen for nuclear expression of changes of the IL-2 gene transcription factors. Our findings revealed that changes in mitogen-stimulated T-cell AP-1 and NFkappaB factor activation correlated directly with defective mitogen-induced IL-2 mRNA expression. We determined that there was a loss of nuclear AP-1 activation and changes in NFkappaB factor activation at 9 days after injury. T-cell nuclear extracts prepared from sham injured mice showed induction of NFkappaB2 (p52) and RelA (p65) containing NFkappaB EMSA complexes, while we detected no RelA or NFkappaB2 in EMSA complexes using T-cell nuclear extracts prepared from burn injured mice. Instead, these NFkappaB EMSA complexes contained mostly NFkappaB1 (p50). Western immunoblot analysis confirmed defective nuclear RelA translocation. Taken together, these results indicate that T-cell NFkappaB and AP-1 activation pathways may be involved in the injury-induced changes in T-cell cytokine production and the immune deviation that occurs after injury.

Immunotherapy with RNA in cancer.

The effect of I-RNA therapy was studied in a B16 melanoma-C57BL/6J mouse system. After having primary B16 isografts excised, mice receiving syngeneic lymphocytes incubated in vitro with specific guinea pig B16 I-RNA showed significantly improved survival as compared to control mice receiving untreated lymphocytes. This therapeutic effect was tumor specific and RNase sensitive. Significant cytotoxicity against B16 cells in vitro was consistently observed with lymphocytes prepared from B16 I-RNA treated animals, whereas lymphocytes from control animals or those treated with RNase-degraded B16 I-RNA or 3LL I-RNA had no effect. Results suggest that the combination of surgery and immunotherapy with I-RNA may be useful in preventing tumor recurrence in certain patients with cancer.

The effects of immunosuppression on the induction and immunogenicity of chemically induced sarcomas.

The induction of chemically induced sarcomas does not appear to be accelerated by immunosuppression with antilymphocyte serum. Tumors induced in immunosuppressed mice contain tumor-specific transplantation antigens of antigenic strength comparable to that of those produced in normal hosts. Transplanted tumors will appear and grow more quickly in ALS-treated mice. Moreover, ALS pretreatment can block the recognition of and response to tumor-specific transplantation antigens.

The fate of bypass grafts to an isolated popliteal artery segment.

From 1967 to 1982, 55 patients underwent 64 femoropopliteal bypass grafts into an isolated popliteal artery segment. Seventy-six percent of these patients had threatened limb loss from advanced atherosclerosis, and 24% had disabling claudication. Forty-five percent of the patients were diabetic. The 30-day operative mortality rate was 1.6%, and the 30-day postoperative amputation rate was 3.2%. Graft potencies were analyzed by the life table method. The 2-year graft patency rate was 70.6%, and the 5-year patency was 60.7%. The 2- and 5-year limb salvage rates were each 83%. With evidence for decreased graft function, four grafts (6%) were successfully revised before failure occurred. Among 10 polytetrafluoroethylene grafts followed up to a maximum of 48 months, there was one early postoperative occlusion, one long-term occlusion, and one early amputation. With respect to patency and limb salvage, the results of isolated popliteal artery segment grafts fall between the 5-year patency and limb salvage rates for autogenous vein grafts to popliteal arteries with at least one tibial vessel runoff (78% patency and 89% limb salvage) and the rates for femoral-tibial/peroneal grafts (5-year patency 56%, limb salvage 69%). An isolated segment is an appropriate recipient vessel for a reconstruction for limb salvage, and reasonably good results can be anticipated.

Anti-interleukin-10 antibody restores burn-induced defects in T-cell function.

BACKGROUND: Studies have shown that susceptibility to sepsis after severe injury correlated with reduced production of T-helper 1 (Th1) cytokines (interleukin-2 [IL-2] and interferon-gamma [IFN-gamma]) and a persistence of T-helper 2 (Th2) cytokines (IL-4 and IL-10). The mechanisms responsible for this effect are not clear. We used a T-dependent antigen to study both the effect of burn injury on antigen-specific Th functions in vivo and the effect of anti-IL-10 antibody on these functions. METHODS: Male A/J mice were anesthetized and given a 25% scald burn or a sham burn. On day 0 all mice were immunized with 100 micrograms trinitrobenzene sulfonic acid (TNP) haptenated ovalbumin (TNP-OVA) in complete Freund's adjuvant. Mice (10 per group) were given 250 micrograms monoclonal rat antimurine IL-10 antibody (anti-IL-10 MAB) or control rat immunoglobin G (IgG) on day 0 and 100 micrograms anti-IL-10 MAB or IgG on day 2. On day 10 the mice were killed to obtain serum and spleen cells. TNP-specific serum antibody isotype titers were determined by enzyme-linked immunosorbent assay (ELISA). Splenocyte proliferation and cytokine-production in response to TNP-OVA or to anti-CD3 MAB were determined by tritiated thymidine incorporation and by ELISA, respectively. RESULTS: Burn injury resulted in depressed levels of the TNP-specific IgG2a antibody isotype (Th1 dependent), whereas TNP-specific IgG1 and IgE (Th2 dependent) levels were not decreased in burn versus sham burn mice. Anti-IL-10 MAB but not IgG restored the IgG2a response. Burn injury also resulted in reduced TNP-OVA-specific proliferation of splenocytes, whereas anti-CD3 proliferation was equivalent in burn and sham mice. TNP-OVA-specific IL-2 and IFN-gamma production were significantly reduced by burn injury. Anti-IL-10 MAB restored TNP-OVA-specific proliferation and antigen-specific IL-2 and interferon-gamma production by splenocytes from burn mice. CONCLUSIONS: Burn injury induces the loss of antigen-specific Th1 cell function, and IL-10 acts as a trigger to down-regulate Th1 activity after injury.

A single dose of endotoxin activates neutrophils without activating complement.

Both complement (C) and neutrophils (PMN) are activated in critically ill patients. To evaluate the role of endotoxin in this response, we studied C activation products and PMN cell surface receptors in seven normal subjects before and after endotoxin (USRef 20 U/kg) or saline solution administered on separate occasions. By 4 hours, with endotoxin only, all subjects had myalgia, headache, an increase in body temperature and heart rate, and leukocytosis that returned to normal by 24 hours. At the same time, PMN cell surface receptors for the complement opsonin C3b increased, as measured by indirect immunofluorescence, rising to 251 +/- 44% of baseline by 4 hours (p less than 0.01) and remaining elevated at 24 hours (237 +/- 16%, p less than 0.01). PMN receptors for iC3b increased to 308 +/- 49% of baseline by 4 hours (p less than 0.02) and returned to normal by 24 hours. There was no change in plasma of C3a desArg, C4a desArg, and C5a desArg (4 hours: mean C3a: 153.4 +/- 11.5 ng/ml versus 176.2 +/- 16.2 ng/ml for saline solution, p = ns; C4a: 159.6 +/- 32 ng/ml versus 151.4 +/- 21 ng/ml, p = ns; C5a: undetectable). To confirm the lack of C activation, we examined PMN chemotaxis (CTX) to C5a for any impairment caused by prior in vivo exposure to C5a. CTX to C5a was unaffected (4 hours: 109% +/- 22% of normal versus 114% +/- 10% for saline solution, p = ns). PMN CTX to formyl-methionyl-leucine-phenylalanine and PMN phagocytosis and killing of S. aureus were also unaffected by endotoxin. Thus, a single dose of endotoxin produced a subjective febrile illness and precipitated sustained PMN activation as indicated by increased PMN cell surface complement receptor number in the absence of C activation.

Neutrophil activation after burn injury: contributions of the classic complement pathway and of endotoxin.

We attempt to elucidate the mechanisms of neutrophil (PMN) activation after burn injury. We previously reported prolonged elevations of PMN cell surface complement (C) opsonin receptor levels after burn trauma with a corresponding period of depressed PMN chemotaxis to C5a, which suggests that the C product, C5a, was responsible for PMN activation. However, a lack of direct correlation of C activation with C receptor levels soon after injury raised the possibility of a second PMN-activating substance. We therefore investigated the effect of endotoxin (LPS) on the expression of the C receptors (CR1 and CR3) by normal human PMNs. Concentrations from 0 to 50 ng/ml of LPS 026:B6 caused a dose response increase in the PMN surface expression of CR1 and CR3 as assessed by monoclonal antibody binding and indirect immunofluorescence. The relative CR1-dependent fluorescence rose from a mean of 50 to 385 and CR3 from 50 to 300. Chelation by ethylenediaminetetra acetic acid (EDTA) did not influence this dose response, thus ruling out the possibility of C activation by LPS--an inference supported by the lack of complement activation observed with these concentrations of LPS in normal serum. A similar dose response was obtained in the absence of other cell types or serum, which implies a direct effect that mimicked that of C5a. To determine the mechanism of the later, prolonged C activation after burn injury, we next examined C activation products in 22 patients with burn injuries. Elevations of plasma C3a desArg were present and persisted for 50 days. Elevations were at maximum levels on days 9 through 13 postburn (mean +/- standard error of mean [SEM], 496 +/- 47 ng/ml versus normal 113 +/- 32; p less than 0.01). These were accompanied by elevations of C4a desArg (917 +/- 154 ng/ml versus normal 424 +/- 50; p less than 0.01), which are indicative of classic pathway activation. Finally, we examined PMN function, phagocytosis and percentage killing of Staphylococcus aureus, and found PMN function to be unaltered in the 22 patients. Thus PMN activation after burn injury appears to be caused by LPS soon after injury and by C5a later after injury and affects only selected PMN functions.

A central role for CD95 (Fas) in T-cell reactivity after injury.

BACKGROUND: Recent findings indicate that severe injury primes the immune system for an enhanced and lethal proinflammatory cytokine response against bacterial-derived superantigens. This study asked whether this response to injury involves the CD95 (Fas) signaling pathway. METHODS: To assess superantigen-mediated mortality, wild-type (WT) C57BL/6 and Fas-deficient C57BL/6 lpr (-/-) (lpr) mice underwent burn or sham injury and were challenged 2 hours later with staphylococcal enterotoxin B (SEB). Spleen cells from sham and burn WT or lpr mice were stimulated in vitro with SEB to assess injury effects on IL-2, TNF-alpha, and IFN-gamma production. RESULTS: Lpr burn mice survived the SEB challenge (100% survival), while WT burn mice showed a high mortality (17% survival, P < 001, analysis of variance [ANOVA]). Sham lpr or WT mice suffered no mortality to the SEB challenge. In vitro studies demonstrated that burn lpr mice produced significantly less TNF-alpha, IFN-gamma, IL-2 than burn WT mice (P <.01, ANOVA). Burn injury markedly enhanced SEB-stimulated IFN-gamma production by WT spleen cells and CD8+ T cells, while this did not occur in SEB-stimulated lpr spleen cells. CONCLUSIONS: These findings support the hypothesis that the CD95 (Fas) signaling pathway plays an integral role in the injury-induced enhanced and lethal T-cell reactivity against bacterial superantigens.

The humoral immune response after thermal injury: an experimental model.

BACKGROUND: Severe thermal injury is associated with major alterations in cell-mediated immunity. Because most B-cell responses are regulated or critically dependent on T-cell help, it is not surprising that many studies have also shown a variety of defects in humoral immunity after thermal injury. However, the nature of the relationship between the in vitro ability to produce antibody and subsequent in vivo responses remains unclear. METHODS: With a murine model of thermal injury, the primary and secondary humoral immune response to tetanus toxoid (TT) was examined during a 6-week period after sham burn or burn injury. Serum anti-TT titers and the numbers of anti-TT-secreting splenocytes were determined. RESULTS: Splenocytes from burned animals displayed normal or decreased TT-specific immunoglobulin (Ig) M plaque formation. In contrast, however, IgG plaque formation was persistently increased for up to 6 weeks after thermal injury, suggesting a switch from IgM to IgG antibody production. Conversely serum titers of TT-specific IgG antibody were persistently lower in burn, compared with sham groups. Changes in serum immunoglobulin levels did not account for this marked discrepancy between enhanced in vitro IgG plaque formation but impaired in vivo levels of TT antibody. CONCLUSIONS: The data suggest that thermal injury is associated with a diminished ability to propagate and maintain a normal IgG antibody response, despite the presence of normal or increased numbers of antigen-specific B cells.