Functional heterogeneity in macrophages activated by Corynebacterium parvum: characterization of subpopulations with different activities in promoting immune responses and suppressing tumor cell growth.

Peritoneal cells (PEC) from mice injected i.p. with heat-killed Corynebacterium parvum (CP) showed enhanced immunostimulatory (accessor or A cell) activity as measured by their ability to restore the immune responsiveness of nonadherent spleen cells to sheep erythrocytes (SRBC) and polymeric flagellin (POL) of Salmonella adelaide in vitro. This was true whether the PEC and nonadherent spleen cells were in direct contact or separated by a cell-impermeable membrane which allowed the free passage of soluble mediators. CP-activated PEC also exhibited greatly increased cytostatic activity against the growth of syngeneic tumor cells in vitro. After fractionation of the PEC according to cell size by velocity sedimentation, a separation of A cell activity from anti-tumor activity was observed. Although both these functions were associated with phagocytic cells of the monocyte-macrophage series, the highest A cell activity was found in fractions containing small and medium-sized macrophages, whereas the anti-tumor activity increased with cell size to a maximum with the largest macrophages. Thus, there is a relative increase of suppressive activity over stimulatory activity with an increase in cell size. Cytochemical and morphologic evidence suggests that the A cell-rich fractions contained small and medium-sized macrophages which were derived from newly arrived monocytes, whereas the large tumor-suppressive macrophages were relatively more differentiated.     

Macrophage heterogeneity in tumor resistance: Cytostatic and cytotoxic activity of corynebacterium parvum-activated and proteose peptone-elicited rat macrophages against Moloney sarcoma tumor cells

Peritoneal macrophages from proteose peptone and Corynebacterium parvum (CP)-treated Lewis and Brown Norway rats were separated into subpopulations by centrifugation on discontinuous gradients of Ficoll. Four macrophage subpopulations were prepared and tested for cytostatic and cytotoxic activity against syngeneic and allogeneic Moloney sarcoma tumor cells. Macrophages were cocultured with tumor cells for 48 hr, whereupon either the inhibition of [¹²⁵I]iododeoxyuridine uptake was measured (cytostasis) or the tumor monolayers were observed for cytotoxic effects. CP-Activated macrophages from heavy-density portions of the gradient (8–10% and 10%-pellet) were highly cytostatic and cytotoxic to both the syngeneic and allogeneic tumor cells while macrophages from the light-density portions (4–6 and 6–8% Ficoll bands) were not. Proteose peptone-stimulated macrophages from the heavy-density portions of the gradient were cytostatic but not cytotoxic to the tumor cells. The effector macrophages from the CP-activated pool were large, well-differentiated cells as determined by electron microscopic examinations and had enhanced phagocytic activity when contrasted with the noncytotoxic, less dense macrophages.     

Synergistic cytolytic activity by combined populations of peritoneal T lymphocytes and macrophages during the L5178Y cell tumor-dormant state in DBA/2 mice

It was previously reported that the establishment of the L5178Y cell tumor-dormant state in DBA/2 mice is mediated principally by a peritoneal cytolytic T-cell response that reaches peak levels 4 days after L5178Y cell challenge, lyses more than 99% but less than 100% of peritoneal L5178Y cells, and gradually wanes to background levels by 40–70 days postchallenge (DPC). At this time the majority of mice are clinically normal, and contain a relatively small number of L5178Y cells in the peritoneal cavity. During the tumor-dormant state, mice that harbor more than 10⁴ L5178Y cells contain peritoneal macrophage-mediated cytolytic activity. We report here that tumor-dormant mice that contain fewer than 10⁴ peritoneal L5178Y cells also produce cytolytic activity in vitro, but that it is synergistic, in that the cytolytic activity of adherent (AD) peritoneal cells (PEC) and nonadherent (NAD) PEC cultured together is greater than the additive lysis produced by these cell populations when cultured separately. This synergistic cytolytic activity is: (1) effector cell density dependent, (2) dependent on the tumor-dormant status of the NAD and AD PEC donor mice, (3) protracted in its kinetics during a 48-hr in vitro assay, and (4) dependent on an interaction between NAD T cells and AD phagocytic macrophages. The consistent detection of this in vitro-assayed cytolytic activity in PEC of tumor-dormant mice which harbor small endogenous tumor burdens suggests that it reflects an in vivo cytotoxic effector mechanism involved in the long-term maintenance of the tumor-dormant state.     

Characterization of the cells that suppress the cytotoxic activity of T lymphocytes. II. Physical properties, specificity, and developmental kinetics of the inhibitory splenic non-T cell population.

The in vivo immune response to alloantigens (tumor allograft reaction and acute graft-versus-host reaction) generates a population of antigen-specific “killer” T cells (CTL) and a separate activity (INA) inhibiting target cell lysis in vitro. Using a simple mathematical model to quantitate each activity from ⁵¹Cr-release titration curves, we show that cells with inhibitory activity differ from CTL in several respects: INA resists destruction by RAMB + C, acts without specificity for the sensitizing alloantigen, and appears to act by a cell-contact mechanism which does not require that the inhibitory cells remain viable. Cells with INA produce noncompetitive inhibition, in contrast to the competitive inhibition produced by alloantigen. Velocity sedimentation separation indicates that INA is quite heterogenous; normal spleen contains low levels of INA, which is associated with small cells (s ? 3 mm/hr). Increased INA develops in a regular manner during an immune response and is associated with a larger inhibitor population (s > 4 mm/hr). Separation on plastic dishes and with carbonyl iron powder demonstrates that INA is not restricted to macrophages.     

Similarities in enhanced glucosamine incorporation by macrophages stimulated with migration inhibitory factor and the fucolectin from Lotus tetragonolobus

Fucose-binding protein (FBP), the fucolectin from Lotus tetragonolobus, was compared with migration inhibitory factor (MIF) for its ability to stimulate [¹⁴C]glucosamine incorporation into trichloroacetic acid (TCA)-insoluble material of guinea pig peritoneal exudate cells (PEC) using a microtiter assay. Both MIF and FBP inhibit macrophage migration and were shown to stimulate glucosamine incorporation in a similar dose response fashion over time. Both unpurified PEC and PEC depleted of nonadherent cells displayed significant levels of glucosamine incorporation when stimulated by MIF or FBP. Tunicamycin and 2-deoxy-d-glucose, known inhibitors of glycosylation, inhibited glucosamine incorporation by control and MIF- or FBP-stimulated PEC. These results confirm the similarities between MIF and FBP in their biological activity for macrophages using a second in vitro correlate of cell-mediated immunity and suggest involvement of enhanced glycoprotein or glycolipid biosynthesis by FBP and lymphokine-activated macrophages.     

Down-regulation of cytotoxic T lymphocyte development by a minor stimulating locus-induced suppressor cascade that involves Lyt-1+ suppressor T cells, IA- macrophages, and their factors

Down-regulation of the development of CTL has been studied in mice both in vivo and in vitro. To generate CTL to hapten-altered self Ag in vivo, an immunization protocol has been used in which the host's Th cells are stimulated by a minor locus histocompatibility Ag (Mlsd) and its precursor CTL are activated by trinitrophenylated syngeneic spleen cells. Injecting the H-2 compatible Mls-disparate spleen cells along with the TNP-coupled self cells into the hind paws causes TNP-self specific CTL to appear in popliteal lymph nodes within 5 days. We have previously reported that inducing Ts cells by i.v. injecting Mlsd-bearing cells prevents in vivo generation of TNP-self specific CTL after immunization in this way. Here the induced Ts cell as well as the mechanism by which it functions have been further examined. The suppression was seen to extend to allogeneic as well as TNP-self Ag, provided the Mlsd-tolerized animal was reexposed to Mlsd-bearing cells at the time of immunization for CTL. By transferring the Mlsd-induced suppression adoptively we have learned that the splenic suppressor cell bears Thy-1.2 as well as Lyt-1.1 Ag and inhibits the generation of CTL at the afferent limb. In addition, Mlsd-induced PEC of Mlsd-tolerized mice, but not of normal mice, mediated suppression of development of CTL in vivo. The active cells within the tolerized PEC have been identified as T cells and macrophages (M phi). Furthermore, PEC from mice tolerized to Mlsd suppressed generation of CTL directed toward TNP-self targets in vitro. T cells and M phi separated from PEC of Mlsd-tolerized mice achieved suppression best in culture when present together. In addition, Lyt-1+ splenic cells from tolerized but not normal mice cooperated to down-regulate CTL generation in vitro with peritoneal M phi from either tolerized or normal mice. Supernatants of 24- to 72-h cultures of PEC from tolerized mice were suppressive of CTL generation when incorporated at 40 to 50% of culture volume. Supernatants of T cells from tolerized PEC or spleen were suppressive in culture only when M phi from normal mice were also present. To achieve suppression dialyzed supernatants of M phi from tolerized mice could replace the M phi.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation and mechanism of action of suppressor macrophages

Intravenous administration of Corynebacterium parvum to alloimmunized mice activates splenic suppressor macrophages that effectively curtail primary and secondary generation of cytotoxic T lymphocytes (CTLs) in vitro. CTL generation was significantly inhibited in suppressed primary cultures by Day 3, the earliest time point that activity is first detected in control cultures. Suppressor macrophages had to be present during the first 24–48 hr of culture to effectively curtail the generation of CTLs. However, if suppressor macrophages were reactivated by 48-hr in vitro culture and then added to primary sensitizations that had been initiated 48 hr previously, they were capable of significant suppression. Suppressor cells produced a soluble factor that mediated the inhibition of CTL generation. The production or action of this factor could not be counteracted by indomethacin.     

Inhibition of the lytic phase of murine T-cell-mediated alloimmune cytotoxicity by a rat antiactivated T-cell antiserum

Antisera produced in rats by immunization with alloimmune murine C57Bl/6 anti-P815 splenic lymphocytes or purified T cells activated in vitro by coculture with phytohemagglutinincoated L-929 cells were found to inhibit the in vitro cytolytic action of in vivo and in vitro alloimmune C57Bl/6 anti-P815 cytotoxic T cells in a 4-hr chromium-51 release assay. The rat anti-murine-activated lymphocyte (anti-MAL) or antiactivated T-cell (anti-ATC) serum inhibited lysis in the absence of exogenously added complement activity and were not directly cytotoxic to CTL. Absorption of anti-MAL with target cells P815, L-929, EL-4, and normal C57Bl/6 lymphocytes removed a limited amount of the CTL-inhibitory activity. In contrast, lectin-activated alloimmune lymphocytes fully absorbed the inhibitory activity indicating these antisera preferentially recognize unique antigenic determinants associated with the activated CTL cell surface. The anti-ATC was found to block alloimmune lysis by CTL from several inbred mouse strains suggesting these antisera recognized antigenic determinants of a common lytic mechanism. A kinetic analysis of the inhibitory activity of the anti-MAL on the CTL reaction scheme revealed this antiserum inhibited lysis at a post-Ca²⁺-dependent step, presumably during the target cell lytic phase. This result suggests the rat antiserum can neutralize the CTL lytic mechanism.     

Adriamycin-induced activation of NK activity may initially involve LAF production

C3HeB/FeJ spleen cells (unseparated or passaged over nylon wool columns) were cultured overnight (1-2 X 10(6) cells/microwell) in the presence and absence of resident or ADM-induced PEC and anti-YAC-1 (4h) NK activity was determined. The addition of resident PEC to spleen cells had little effect on NK activity. However, the addition of ADM-elicited PEC (10 mg/kg, IP, day -1 and day -5) to spleen cells prior to culture significantly augmented NK activity. If ADM-induced PEC were treated with carbonyl iron prior to coculture with spleen cells, augmentation of anti-YAC-1 activity was not observed. This suggested that ADM-activated macrophages augmented cultured splenic NK activity. Supernatants from overnight-cultured resident or ADM-induced adherent PEC were then prepared, dialyzed (to remove ADM), and tested for mitogenic activity or cocultured with spleen cells overnight. ADM-induced adherent PEC supernatants stimulated the proliferation of murine thymocytes (both LAF and IL-2 also stimulate) but not cultured CTL (only IL-2 stimulates). ADM-induced adherent PEC supernatants (as well as LAF, IL-2, and IFN) augmented overnight-cultured C3HeB/FeJ splenic NK activity. However, only IL-2 and IFN could augment overnight-cultured athymic BALB/c . nu/nu splenic NK activity. This suggested that ADM-elicited macrophages produce LAF which may act directly on NK cells or, more likely, may induce T cells to produce IL-2, IFN, or both.     

Inhibition of the mixed lymphocyte culture by peritoneal exudate cells.

Inhibition of mixed lymphocyte cultures (MLC) by macrophages and by supernatants of short term cultured macrophages was assessed by incorporation of ³H-thymidine (TdRH3) and also by blast cell counts and by determination of cellmediated lympholysis. Peritoneal exudate cells (PEC) induced by thioglycollate, at concentrations >10%, inhibited all three parameters of MLC. Lower concentrations of PEC, and supernatants from cultured PEC, inhibited TdRH3 incorporation, but had no significant effect on blast cell counts or on generation of cytotoxic effector cells. Inhibition by the supernatants could be reversed by dialysis or by use of low specific activity TdRH3. These data indicate that macrophages can inhibit proliferative responses in MLC, but that this must be carefully distinguished from selective inhibition of TdRH3 incorporation.     

Antigen-specific murine T-cell proliferation: role of macrophage surface Ia and factors.

The proliferation of Mycobacterium-primed murine lymph node T cells to purified protein derivative of tuberculin (PPD), as measured by the uptake of tritiated thymidine, requires the obligatory participiation of macrophages which stimulate the T cells either directly with antigen in association with cell surface Ia (I region-defined antigens), or indirectly by means of soluble factors. We have examined the possibility that this functional dichotomy is due to heterogeneity within the macrophage population. Since the maturation of macrophages from the precursor monocytes is associated with cell enlargement, macrophage subpopulations differing in developmental stage are obtained by cell fractionation according to size by velocity sedimentation. Nylon-wool-purified T cells which have been depleted of macrophages and B cells are stimulated with PPD either in a free form or bound to macrophages which have been incubated for a short time (i.e., pulsed) with PPD. We found that for PPD-pulsed macrophages, only the smallest (and probably the most immature) are capable of inducing T-cell proliferation. This antigen presentation function is mediated by cell surface Ia since it is abolished by pretreatment of the macrophages with anti-Ia serum and complement. On the other hand, all macrophages, irrespective of sensitivity to anti-Ia serum, secrete factors which will stimulate T-cell proliferation in the presence of free PPD. Thus the maturation of macrophages is accompanied by a shift from Ia-dependent to Ia-independent mechanisms of immunostimulation.     

Effects of the MER tubercle bacillus fraction on the production of antibodies in vitro: I. Effect on the primary response

The effect of the methanol extraction residue (MER) fraction of BCG tubercle bacilli on the primary antibody response in vitro to sheep red blood cells (SRBC), TNP conjugates, and the monovalent hapten DNP-glycine was studied. Addition of MER to whole splenocyte cultures simultaneously with antigen presentation potentiated the antibody response to SRBC and TNP-SRBC, and facilitated reactivity to DNP-glycine; there was no effect on the response to the T-independent entity TNP-LPS (lipopolysaccharide from E. coli 055-B5). Immunopotentiating activity of MER for SRBC and DNP-glycine was also evident in macrophage-depleted cultures. Peritoneal exudate cells (PEC) taken from MER-treated donors were more efficient than PEC from untreated donors in reconstituting antibody formation to SRBC by macrophage-depleted spleen cell populations. The results obtained indicate that activation of both macrophages and of certain lymphocyte population(s) by MER may play a role in the potentiation of antibody responsiveness in vitro by this agent.     

Cyclophosphamide modifies the induction kinetics but not cell types and cytotoxic mechanisms of antitumor cells elicited with OK-432 plus attenuated tumor cells

Summary The present study was designed to examine whether the antitumor cells induced by treatment with mitomycin-C-treated EL4 cells (EL4_MMC) plus OK-432 plus cyclophosphamide differed from those induced by treatment with EL4_MMC plus OK-432 in terms of their cell types and antitumor mechanisms. Antitumor activity of peritoneal exudate cells (PEC) from mice receiving either treatment was nonspecific, and inhibition of their target cell growth increased for up to 24 h. Macrophage toxin, silica and trypan blue abrogated the activity in vitro and in vivo, respectively. The activity of the PEC was inhibited with inhibitors of the arachidonic acid cascade, such as dexamethasone, 4-bromophenacyl bromide and nordihydroguaiaretic acid but not esculetin, ibuprofen, indomethacin and BW755C.N ^G-monomethyl-l-arginine, a specific competitive inhibitor of thel-arginine-dependent nitric oxide synthesis, also inhibited the activity. These results and morphological observations indicated that antitumor cells in the PEC from mice receiving either treatment were macrophages, and that their activity was closely related to the arachidonic acid cascade and to nitric oxide. Antitumor activity of the PEC spontaneously decayed in vitro and this decay was inhibited by the addition of OK-432 or lipopolysaccharide. On the other hand, cyclophosphamide sustained the appearance of antitumor cells in mice treated with EL4_MMC plus OK-432. Therefore, cyclophosphamide treatment did not modify cell types and cytotoxic mechanisms of antitumor cells elicited with EL4_MMC plus OK-432, but did modify the induction kinetics of such antitumor macrophages.     

Role of the H-2I region in the generation of an antiviral cytotoxic T-cell response in vitro

The participation of H-2I gene products in generating virus-specific proliferative and/or cytotoxic T-lymphocyte (CTL) responses was investigated. Spleen cells from mice infected with vaccinia virus were restimulated secondarily in vitro with syngeneic virus-infected peritoneal exudate cells (PEC) and then restimulated in tertiary cultures with virus-infected PEC from syngeneic and partially histoincompatible strains of mice. Based on the finding that comparable proliferative responses resulted when stimulating the responding cells were histocompatible at the H-2K, I, or D region of the major histocompatibility complex (MHC), the additively enhanced, but not potentiated, proliferation caused by identity at two or three H-2 regions was analyzed. Enhancement of proliferation followed when the H-2K/D components plus virus and the H-2I components plus virus were either on the same, or alternatively on two, stimulating cells. This suggests that H-2K, D, and I plus virus trigger distinct T-cell subsets. A virus-specific CTL response was generated in vitro when spleen cells from virus-primed mice and even unprimed mice were stimulated with cells sharing only H-2K and/or H-2D of the MHC. Identity at the H-2I region did not stimulate a CTL response, nor did it influence the magnitude of the $\text{K}\text{D}$ restricted response. Nevertheless, the presence of anti-Ia antiserum in cultures of syngeneic stimulators and responders inhibited the cytotoxic response to a great extent. Therefore, H-2I region products seem to participate in the generation of virus-specific CTL in vitro.     

CD40 ligation restores cytolytic T lymphocyte response and eliminates fibrosarcoma in the peritoneum of mice lacking CD4+ T cells

Absence of CD4⁺ T cell help has been suggested as a mechanism for failed anti-tumor cytotoxic T lymphocytes (CTL) response. We examined the requirement for CD4⁺ T cells to eliminate an immunogenic murine fibrosarcoma (6132A) inoculated into the peritoneal cavity. Immunocompetent C3H mice eliminated both single and repeat intraperitoneal (IP) inoculums, and developed high frequency of 6132A-specific interferon-γ (IFNγ)-producing CTL in the peritoneal cavity. Adoptive transfer of peritoneal exudate cells (PEC) isolated from control mice, protected SCID mice from challenge with 6132A. In contrast, CD4 depleted mice had diminished ability to eliminate tumor and succumbed to repeat IP challenges. Mice depleted of CD4⁺ T cells lacked tumor-specific IFNγ producing CTL in the peritoneal cavity. Adoptive transfer of PEC from CD4 depleted mice failed to protect SCID mice from 6132A. However, splenocytes isolated from same CD4 depleted mice prevented tumor growth in SCID mice, suggesting that 6132A-specific CTL response was generated, but was not sustained in the peritoneum. Treating CD4 depleted mice with agonist anti-CD40 antibody, starting on days 3 or 8 after initiating tumor challenge, led to persistence of 6132A-specific IFNγ producing CTL in the peritoneum, and eliminated 6132A tumor. The findings suggest that CTL can be activated in the absence of CD4⁺ T cells, but CD4⁺ T cells are required for a persistent CTL response at the tumor site. Exogenous stimulation through CD40 can restore tumor-specific CTL activity to the peritoneum and promote tumor clearance in the absence of CD4⁺ T cells.Supported in part by grants from Children’s Hospital of Wisconsin Foundation, Society of University Surgeons Foundation, Florence and Marshall Schwid Foundation, Elsa Pardee Foundation, Kathy Duffy Fogarty Fund of the Greater Milwaukee Foundation (JS) and NIH grant RO1-CA-37156 (HS); Andrew Lodge and Ping Yu have contributed equally to this work.     

Antibody-dependent murine macrophage-mediated damage to Schistosoma mansoni schistosomula in vitro

Antibody-dependent cell-mediated cytotoxicity (ADCC) against schistosomula of the human parasite Schistosoma mansoni was demonstrated using antisera from mice plus peritoneal exudate cells (PEC). PEC were divided into plastic-adherent (96% macrophages, 4% lymphocytes) and nonadherent (92% lymphocytes, 8% macrophages) cell populations. Four criteria of ADCC were used, including minimal and maximal cell attachment, and death of and uptake of trypan blue by schistosomula. Using cells from normal mice and antisera from schistosome-infected mice, macrophages adhered to, damaged the tegument and underlying structures of, and killed schistosomula when observed following 18 hr incubation. In homologous systems, the results were similar when outbred CD-1 and inbred BALB/c mice were compared, except that potency of antisera from the latter mice decreased after 6–7 weeks postinfection, whereas the opposite was true for the former strain of mice. Nonadherent cells also exhibited ADCC against schistosomula, but the potency was considerably lower than that of adherent cells. These complement-independent ADCC reactions were stage-specific for the schistosomulum in that no reactions occurred with adult worms.     

Mycobacterium tuberculosis Multidrug Resistant Strain M Induces an Altered Activation of Cytotoxic CD8+ T Cells

In human tuberculosis (TB), CD8⁺ T cells contribute to host defense by the release of Th1 cytokines and the direct killing of Mycobacterium tuberculosis (Mtb)-infected macrophages via granule exocytosis pathway or the engagement of receptors on target cells. Previously we demonstrated that strain M, the most prevalent multidrug-resistant (MDR) Mtb strain in Argentine, is a weak inducer of IFN-γ and elicits a remarkably low CD8-dependent cytotoxic T cell activity (CTL). In contrast, the closely related strain 410, which caused a unique case of MDR-TB, elicits a CTL response similar to H37Rv. In this work we extend our previous study investigating some parameters that can account for this discrepancy. We evaluated the expressions of the lytic molecules perforin, granzyme B and granulysin and the chemokine CCL5 in CD8⁺ T cells as well as activation markers CD69 and CD25 and IL-2 expression in CD4⁺ and CD8⁺ T cells stimulated with strains H37Rv, M and 410. Our results demonstrate that M-stimulated CD8⁺ T cells from purified protein derivative positive healthy donors show low intracellular expression of perforin, granzyme B, granulysin and CCL5 together with an impaired ability to form conjugates with autologous M-pulsed macrophages. Besides, M induces low CD69 and IL-2 expression in CD4⁺ and CD8⁺ T cells, being CD69 and IL-2 expression closely associated. Furthermore, IL-2 addition enhanced perforin and granulysin expression as well as the degranulation marker CD107 in M-stimulated CD8⁺ T cells, making no differences with cells stimulated with strains H37Rv or 410. Thus, our results highlight the role of IL-2 in M-induced CTL activity that drives the proper activation of CD8⁺ T cells as well as CD4⁺ T cells collaboration.     

Induction of ornithine decarboxylase in macrophages by bacterial lipopolysaccharides (LPS) and mycobacterial cell wall material

Lipopolysaccharide from $\text{E. Coli}$ (LPS) and BCG cell walls (BCGcw) are recognized immunoadjuvants that directly stimulate some macrophage functions. The macrophage cell line J774.1 and peritoneal exudate cells (PEC) from mice can be stimulated by LPS or other adjuvants $\text{in vitro}$ to synthesize and release protein factor(s) that activate thymus-derived lymphocytes. We have utilized J774.1 cells and PEC to demonstrate that an increase in ornithine decarboxylase (ODC) activity is a marker of early biochemical changes in adjuvant-stimulated macrophages. BCGcw and LPS increased ODC within 2 hours in J774.1 cells as well as murine peritoneal exudate macrophages. Maximal increases in ODC were detected 4 hours after the addition of adjuvants to J774.1 cells. The marked increases (12–23 fold) in ODC observed with BCGcw (20 μg/ml) did not appear to involve an effect on cell proliferation which was suppressed by this adjuvant. Cycloheximide inhibited the induction of ODC by LPS and BCGcw in the macrophage cell line. Evidence that the induction of ODC may be promoted by an increase in cyclic AMP was provided by experiments demonstrating that prostaglandin E₁ (PGE₁) and 8-bromo-adenosine-3′:5′-monophosphate (8Br-cyclic AMP) can mimic the effects of LPS and BCGcw in J774.1 cells. These observations indicate that one of the early biochemical changes in macrophages promoted by adjuvants is an induction of ODC.     

In situ effector pathways of allograft destruction. 1. Generation of the "cellular" effector response in the graft and the graft recipient

Inflammatory leukocytes of DA-to-WF rat renal allografts displayed significant cytolytic activity to natural killer (NK) target cells on Day 2 after transplantation. The NK activity, which was associated with large granular lymphocytes in discontinuous Percoll gradients, peaked on Day 4 and disappeared rapidly thereafter. Coincident with the presence of NK activity in the graft, a decrease in NK activity in the recipient spleen was observed. Low NK activity was also recorded in WF-to-WF autografts. The cells displaying direct cytotoxic activity to donor (but not to recipient) strain peritoneal exudate target cells (PEC) were associated with the T suppressor/killer lymphocytes in affinity chromatography. They appeared in the graft between Days 2 and 4, peaked between Days 6 and 8 and disappeared slowly thereafter. In the spleen the cytotoxic T lymphocyte (CTL) activity appeared later and it reached a maximum between Days 16 and 20 before decreasing. In the blood distinct CTL activity was seen only from Days 16-20 onwards, after the graft had been rejected. No CTL activity was recorded in the graft, blood, or spleen of an autograft recipient. Addition of donor-directed post-transplantation antibody (antibody-dependent cellular cytotoxicity, ADCC) had a slight enhancing effect on the cytotoxic activity of inflammatory leukocytes up to Day 5. After this time, added antibody had a blocking effect on direct CTL activity. No ADCC activity was recorded in the inflammatory population of an autograft. On the contrary, high levels of ADCC activity to donor strain PEC were recorded in the spleens of both autograft and allograft recipients throughout the period of follow-up. The results demonstrate that at least three cellular effector pathways exist in an allograft: a strong natural killer cell component, a strong cytotoxic T lymphocyte component, and (possibly) a weak cell component participating in an ADCC type of cytotoxicity.     

Natural product pectolinarigenin exhibits potent anti-metastatic activity in colorectal carcinoma cells in vitro and in vivo

Colorectal carcinoma (CRC) is one of the most common cancers with high metastatic potential, explaining why identifying new drug candidates that inhibit tumour metastasis is an urgent need. The aim of this study was to evaluate the biological activities of pectolinarigenin (PEC, a natural flavonoid present in Cirsium chanroenicum) in CRC in vitro and in vivo and to determine its underlying mechanism of action. Here, we observed that treatment with PEC could inhibit cell viability and induce apoptosis in cancer cells in a concentration- and time-dependent manner. The occurrence of apoptosis was associated with activation of caspase-3 and Bax and decreased expression of Bcl-2. In addition, PEC markedly impaired CRC cell migration and invasion by downregulating the expression of matrix metalloproteinase (MMP-9) and phosphorylated-Stat3^Tyr705. Moreover, our studies showed that PEC inhibited abdominal metastasis models of murine colorectal cancer. In addition, histological and immunohistochemical analyses revealed a decrease in Ki67-positive cells, MMP9-positive cells and p-Stat3^Tyr705 cells upon treatment with PEC compared to control samples. Furthermore, PEC reduced the number of myeloid-derived suppressor cells (MDSCs) in the blood and tumours, which was accompanied by the increased infiltration of CD8⁺T cells in the blood. Taken together, our findings suggested that PEC could be used as a natural drug to inhibit CRC metastasis.