Peritumoral administration of X63-m-IL-2 cells, transformed by IL-2 cDNA and constitutively producing large quantities of IL-2, was effective in mediating regression of X63-Ag8.653 plasmacytomas transplanted in syngeneic mice. Injection of killer cells with LAK activity substantially enhanced the tumor-inhibitory effect of IL-2-producing cells. In vitro activation of murine bone marrow and spleen cells with IL-2 produced by the X63-m-IL-2 cell line resulted in generation of lymphocytes with oncolytic activity against X63-Ag8.653 plasmacytomas and a variety of other target cell lines. The cytolytic activity of adherent IL-2-activated killer cells (A-LAK) was substantially higher than that of a nonadherent subset. Bone marrow and spleen cells from both healthy control and tumor-bearing mice contained LAK precursors, but the cytolytic activity derived from tumor-bearing mice was lower than that from healthy controls.
The effect of sex hormones on concanavalin A (Con A)-activated human T cells was studied. We show that neither 17-beta-estradiol (E2) nor progesterone, in concentrations of up to 10(-6) M, alters the proliferative response of peripheral-blood mononuclear cells (PBMC) of healthy postmenopausal women. Furthermore, the hormones had no effect on the composition of T cell populations and on the expression of activation markers. We extended our study to a unique T cell population that is characterized by the ability to form rosettes with human erythrocytes, following Con A activation (designated autorosette-forming cells; ARFC) and known to manifest suppressive activity. Indeed, the in vitro addition of E2 (neither progesterone nor testosterone) to Con A-stimulated PBMC brought an about 2- to 4-fold increase in the frequency of ARFC. Tamoxifen, an antiestrogen drug, reduced the frequency of estrogen-stimulated ARFC to the original low level. Furthermore, the inhibitory effect of growth medium from ARFC cultures originally stimulated with Con A + E2 was found to be higher than that of ARFC cultures originally stimulated with Con A alone.
In order to determine whether lymphocytes with therapeutic potential can be obtained from colon carcinoma (C26)-bearing (TB) mice, splenocytes were activated either in mixed lymphocyte-tumor cell (MLTC) cultures in the presence of 5 and 10 U/ml IL2 or 250 and 100 U/ml IL2 (LAK effectors). The therapeutic efficacy, as well as functional and phenotypic features of such lymphocytes, was then compared in an adjuvant immunotherapy setting. Comparisons of the antigenic phenotype, cytotoxic and proliferative activities, and of transcription of different cytokine genes (IFN-gamma, TNF-alpha, IL4, IL6) between lymphocytes activated in MLTC and LAK failed to reveal major differences. However, the in vitro lysis of C26 by MLTC-activated but not by LAK TB lymphocytes was significantly blocked by anti-T3 and anti-Lyt2 monoclonal antibodies, suggesting that a fraction of specific anti-tumor effectors was present in the MLTC bulk population. Moreover, the amount of IFN-gamma (but not of other cytokines) produced by MLTC-derived lymphocytes after stimulation with C26 cells was shown to be 10-fold higher than that produced by LAKs. When combined with low-dose IL2 administration as an adjuvant treatment in C26-operated mice, MLTC effectors showed a higher therapeutic activity than LAKs obtained from the same pool of lymphocytes from TB donors. In the same setting, MLTC-activated lymphocytes obtained from TB or tumor-excised (TE) mice, combined with IL2, were equally effective (76 and 74% survivors, respectively, vs. 27% of the surgery control group and 26% of the group given IL2 only), whereas LAK cells from TE but not from TB animals resulted in the cure of a significant fraction of mice.
Deficient cellular cytotoxic mechanisms are present in neonates, contributing to their increased susceptibility to certain viruses, notably herpes simplex virus (HSV). Significant lymphokine-activated killer (LAK) cell activity has been described in cord blood, suggesting a possible role for LAK and/or interleukin-2 (IL-2) therapy in newborns with serious viral infections. The effect of HSV (type 1) on the activation of cord versus adult LAK cells was investigated by adding virus (multiplicity of infection, MOI = 10) to cells that had been previously incubated for 4-6 days with IL-2 (50-100 U/ml). The cells were then tested 24 h after virus exposure for cytotoxic activity against 51Cr-labelled K562 and Raji target cells. HSV inhibited LAK cytotoxicity of adult cells against K562 by 44% (72 +/- 2.4%, SEM; specific lysis to 40 +/- 6.2%, n = 15) and by 62% against Raji targets (50 +/- 5.6 to 19 +/- 4.4%). A similar degree of inhibition was observed for cord cells against K562 (76 +/- 2.0 to 46 +/- 5.3%) and Raji (60 +/- 4.6 to 24 +/- 6.2%). The degree of inhibition was correlated with the dose of virus in dose-response experiments. Inhibition was also noted with irradiated (10,000 rad) but not with heat-inactivated (56 degrees C for 60 min) virus. No inhibition was found when virus was added directly to the cytotoxic assay or when virus was added at the initiation or end of culture of cells with IL-2 (i.e. day 0 or day 5-7). In contrast, HSV stimulated cytotoxic activity against both the natural killer (NK)-sensitive (K562) and NK-resistant (Raji) targets in cells not incubated with IL-2. The cytotoxicity of adult cells incubated with infectious HSV (MOI = 10) for 5-7 days increased from 5.5 +/- 1.9% in the absence of virus to 25 +/- 6.0% against K562 in the presence of virus and from 3.5 +/- 1.0 (no virus) to 16 +/- 4.3% (with virus) against Raji targets (n = 8). The cytotoxicity of cord cells was also stimulated, but to a lesser degree. Irradiated virus also stimulated cytotoxic activity but to a lesser degree in cord cells. Virus-induced nonspecific cytotoxicity may represent an important component of the host's antiviral defense that is present at birth, but somewhat diminished compared to normal adults.
Natural antibodies (NAb), natural killer (NK) cells and activated macrophages have all been implicated in the rejection of threshold syngeneic tumor inocula. Previous analysis of tumor susceptibility in normal versus inbred and F1 mice bearing the B cell deficiency associated with the xid mutation of CBA/N mice demonstrated an inverse relationship between the tumorigenicity of the RI-28, a radiation-induced leukemia of the CBA/H strain, and the pooled anti-RI-28 serum NAb levels in mice with the same genetic origins. No relationship with tumor susceptibility was seen with NK cell or in vivo activated macrophage cytolysis. Flow-cytometric determination of antitumor serum NAb bled from individual male and female (CBA/N X CBA/J)F1 mice 1 week prior to the threshold tumor inoculation has revealed extensive heterogeneity within the NAb levels of each sex. A comparative analysis of tumor fate with NAb activity revealed that tumors appeared in only 26.3% of animals with a mean fluorescence channel binding above 60 channels in contrast with 77.3% of animals with lower NAb levels. These data extend to the level of individual hosts the support for an inverse relationship between host NAb activity and tumor susceptibility. In addition, subsequent analysis of serum antitumor NAb levels, splenic NK cytolysis and in vitro lymphokine-activated macrophage activity with all three mediators originating from the same individual F1 mice showed no consistent correlations between these natural resistance activities, arguing for the exclusion of deficiencies in NK cell or macrophage function as the basis for the differential tumor susceptibility in individual F1 mice.
Peritoneal macrophages (PM) play an essential role in the pathogenesis of bacterial peritonitis, the main complication of peritoneal dialysis (PD). We determined the antibacterial activity of PM from 31 PD patients using gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and gram-negative (Escherichia coli, Pseudomonas aeruginosa) test organisms. In an 8-hour test assay, PM revealed the highest antibacterial activity against E. coli [median bactericidal index (Bi) = 5.46 representing 0.74 log growth inhibition compared to controls] and the lowest against P. aeruginosa (Bi = 1.63, 0.21 log growth inhibition, p less than 0.05). The antibacterial activity against S. aureus (Bi = 1.99, 0.3 log growth inhibition) and S. epidermidis (Bi = 2.0, 0.31 log growth inhibition) was within this range. When compared to peripheral blood polymorphonuclear leukocytes, PM reached only 4% (S. aureus) and 8.1% (E. coli) of their antibacterial activity (p less than 0.05). Using E. coli as a test organism, PM isolated after a 4-hour dialysis period revealed the highest antibacterial activity when compared to PM isolated after longer dialysis periods (p less than 0.05). Increasing the duration of PD to 6 and 8 h subsequently decreased the antibacterial activity of PM, suggesting that unphysiologic concentrations of toxic metabolites in the peritoneal effluent might have a harmful influence on PM functions.
The relationship between depression of early protection against influenza virus infection and the decrease in the number of peripheral polymorphonuclear leukocytes in cyclophosphamide-treated mice was investigated by means of a novel synthetic compound, Y-19995 [2,4'-bis(1-methyl-2-dimethyl-aminoethoxyl)-3-benzoylpyridine dimaleate], which had been shown to exert a potent restorative effect on leukocytopenia in immunocompromised hosts. Following intranasal inoculation with influenza virus (1.5 x 10(3) plaque-forming units) into untreated mice, the pulmonary virus titer progressively increased during 3 days and decreased gradually from day 7 after infection. The treatment with cyclophosphamide 2 days before infection markedly enhanced the pulmonary virus multiplication from the early phase of infection, and the higher virus titer was maintained thereafter. When mice were given Y-19995 after cyclophosphamide treatment, virus titers from the early to late phases of infection were lower than those in untreated mice. The number of peripheral polymorphonuclear leukocytes in cyclophosphamide-treated mice rapidly decreased and returned to normal levels only 9 days after the treatment, while such leukocytopenia was prevented to some extent and the leukocyte count was restored completely up to 7 days by postcyclophosphamide treatment with Y-19995. Furthermore, the treatment with Y-19995 augmented the inactivation of virus by the polymorphonuclear leukocytes. However, the virus inactivation by alveolar macrophages was modified only slightly by Y-19995 treatment. In addition, Y-19995 treatment could potentiate antibody-dependent cell-mediated cytotoxicity of polymorphonuclear leukocytes against the virus-infected target cells, and the production of serum neutralizing antibody to influenza virus in untreated and cyclophosphamide-treated mice. Y-19995 revealed neither antiviral nor interferon-inducing activities.(ABSTRACT TRUNCATED AT 250 WORDS)
IL-2-activated lymphocytes (LAK cells) show increased adherence to, and killing of, human vascular endothelial cells compared to resting lymphocytes. In the present work, we have found that supernatants from LAK cell cultures also are toxic to human umbilical vein endothelial cells (HUVEC) when tested for 48 h in a neutral red uptake assay. Recombinant TNF-alpha and IFN-gamma at high concentrations are also toxic under the same test conditions, and TNF-alpha was directly detected in LAK cell supernatants. An inconsistent inhibition of toxicity was found with anti-TNF-alpha whereas anti IFN-gamma-antibodies had a partial inhibitory effect. The susceptibility of HUVEC to cellular killing by LAK cells could be up- and down-regulated with insulin-like growth factor I and IFN-gamma, respectively. It is concluded that damage to vascular endothelium during high dose IL-2 treatments may be partially related to an excessive production of lymphokines such as IFN-gamma and TNF-alpha. IFN-gamma may, in addition, be protective for HUVEC during cellular interactions with LAK cells.
In this report, we have established that natural killer (NK) cells can increase IgG2a secretion by B lymphocytes as well as alter the distribution of the remaining immunoglobulin isotypes. The effect of NK cells on B cell differentiation is similar to that obtained by the direct addition of recombinant interferon-gamma (IFN-gamma) and, therefore, most likely results from the elaboration of IFN-gamma by NK cells, this is a clear demonstration that NK cells can regulate cell function(s) via a mechanism other than cytotoxicity. In addition, we have shown that the induction of NK cells by B lymphocytes requires close interactions between the two cell types. Further, while only low-density B lymphocytes activated in vivo are effective inducers of NK cells, high-density, resting B cells can be rendered effective by preactivation with either interleukin-4 or anti-mu.
The differentiation of monocytes and macrophages both in vitro and in vivo can be characterized by the modulation of specific functional and molecular phenotypes. We have determined in human peripheral-blood monocytes (HPBM) the role of in vitro differentiation on the expression of nonspecific tumoricidal activity, induction of soluble tumor necrosis factor (TNF) activity and TNF-specific mRNA transcription. HPBM were activatable by bacterial lipopolysaccharide (LPS; 1 microgram/ml) for nonspecific cytotoxicity to A375M (human malignant melanoma cell line) only during the first 24 h of in vitro differentiation. Activated supernatants of HPBM were found to be partially neutralizable (75 +/- 7%) by rabbit polyclonal anti-TNF antibody and, in freshly isolated HPBM, the release of soluble TNF activity determined by the L929 assay was found to occur only after activation with LPS. Maximal TNF release occurred at 8 h of LPS stimulation, and required both protein and RNA synthesis as evidenced by the ability of both actinomycin D and cycloheximide to inhibit its release. Neither control untreated HPBM nor recombinant interferon-gamma (rIFN-gamma; 1 U/ml)-treated HPBM alone released soluble TNF activity. Further in vitro culture determined that HPBM were activatable for TNF release out to 72 h of culture after which HPBM became resistant to LPS-mediated TNF release. The expression of TNF and c-fos mRNA was also determined during in vitro differentiation. Both TNF and c-fos mRNA were expressed in freshly isolated HPBM, and returned to baseline by 24 h of in vitro culture. Treatment of HPBM with LPS induced TNF transcription as late as 5 days of in vitro culture with maximal induction occurring during the first 48 h. rIFN-gamma significantly induced TNF transcription at 24 h in the absence of soluble TNF activity, but did not increase transcription at later times. The expression of nonspecific cytolytic activity, the release of soluble bioactive TNF and the induction of TNF and c-fos mRNA are regulated in HPBM by differentiation-determined processes.
A number of recent studies have shown that mouse target cells (TC) of hematopoietic origin, when exposed to cytotoxic lymphocytes, undergo double-stranded DNA fragmentation. The cause and relevance of the fragmentation remain controversial. In this study we generated a number of mouse (M-LAK) and human LAK (H-LAK) cells and exposed them to a variety of mouse and human TC. YAC and SP/2, 2 mouse TC underwent rapid and extensive fragmentation when lysed by either human or mouse LAK whereas K562 and Daudi, 2 human TC, under the same conditions did not. All 4 TC, however, were killed quite efficiently. Next we labeled TC with 125I-deoxyuridine, exposed them to LAK cells for up to 18 h and loaded the LAK:TC mixtures over an alkaline linear sucrose gradient. After lysing the cells with a lysis buffer containing Triton X-100 we showed that K562 that had been in contact with LAK cells for more than 1 h exhibited single-strand nicks. However, whereas double-strand fragmentation preceded chromium release (lytic activity), the appearance of single-strand nicks did not. Finally, protein synthesis was not required for either type of fragmentation. In summary, we have demonstrated that: (1) the ability to undergo DNA fragmentation is a property of the TC rather than the effector cells that mediated their death, and (2) K562 and Daudi, 2 human TC, undergo single-strand nicks when lysed by LAK cells whereas SP/2 and YAC, 2 mouse TC undergo double-strand fragmentation when exposed to the same syngeneic or xenogeneic effector cells.
Supernatants collected from cisplatin-treated macrophages demonstrated enhanced cytotoxicity against actinomycin-D-treated L929 cells and also enhanced the thymocyte proliferation in response to concanavalin A, showing that cisplatin-treated macrophages release interleukin-1 (IL-1) and tumor necrosis factor (TNF) into the culture supernatant. The supernatant collected from untreated macrophages showed little TNF and IL-1 activity. The release of TNF and IL-1 was observed to be dependent on the dose and duration of cisplatin treatment. Medium alone containing cisplatin did not enhance thymocyte proliferation and had little cytotoxic effect on actinomycin-D-treated L929 cells. Cisplatin-treated macrophage culture supernatants were chromatographed over a Superose 12 column on an FPLC system. TNF activity eluted in two major peaks with apparent molecular weights of 50-55 and 15-20 kilodaltons, respectively. The kinetics of IL-1 release was also studied. Maximum production and release of IL-1 were observed up to 24 h after cisplatin treatment and then gradually declined. Freeze-thaw lysates of cisplatin-treated macrophages also showed enhanced IL-1 activity. Paraformaldehyde (PFA)-fixed cisplatin-treated macrophages showed significantly enhanced cytotoxic activity against L929 cells as compared to PFA-fixed untreated macrophages. PFA-fixed cisplatin-treated macrophages also enhanced thymocyte proliferation. These results suggest that cisplatin treatment of murine macrophages also results in increased expression of membrane-associated IL-1 and TNF activity.
We found that the sera obtained from patients with acute leukemia (AL) in complete remission or malignant lymphoma (ML) during the recovery phase after chemotherapy completely inhibited the GCT-CM-stimulated growth of allogenic and autologous bone marrow colonies in vitro. We investigated 5 AL patients and 6 ML patients from whom blood samples were taken either every day or every 6 h during the recovery phase after chemotherapy. Colony-inhibitory sera, were detected in all patients, more frequently when the peripheral leukocyte count recovered to about 2,500 x 10(6)/l, but then transiently and at intervals. The colony-inhibitory factor purified from the inhibitory sera inhibited the growth of G-CSF-responsive colonies in a dose-dependent manner, but no effect on the growth of GM-CSF-responsive colonies, CFU-E, or BFU-E, was observed. These results suggest that this factor may play a role in regulating granulopoiesis by inhibiting the differentiation of G-CSF-responsive precursor cells.
We report herein that defective natural killer (NK) cell cytotoxicity, NK cytotoxic factor (NKCF) production and NK target binding ability of patients with chronic myelogenous leukemia (CML) are functionally restorable after short-term culture (less than 1 week) with recombinant interleukin-2 (rIL-2). We have previously reported that, despite normal to increased numbers of CD16+ large granular lymphocytes, fluorescence-activated-cell-sorted NK cells from CML patients are profoundly defective in NK cell activity and are unable to lyse the CML blast-crisis-derived, NK-sensitive target K562. Since we and others have also previously shown that the defective NK cytotoxicity from CML patients is restorable after 1-4 weeks of incubation with rIL-2, we therefore deemed it important to study the kinetics of IL-2-mediated NK restoration at earlier time intervals (less than 1 week). In the present report, we have demonstrated a significant restoration of NK cell cytotoxicity in CML patients against K562 after 5 days of short-term culture with rIL-2. In addition, recovery of NKCF production and restoration of target-binding capacity to normal levels by NK cells from CML patients were also observed after short-term (less than 1 week) rIL-2 treatment. Finally, we have demonstrated in the present report that adherent cells and peripheral-blood lymphoid cells from CML patients, as compared to normal controls, are unable to produce IL-1 beta and interferon-gamma, respectively, after stimulation with phorbol myristate acetate (IL-1 beta) and phytohemagglutinin-M (interferon-gamma).
Contact with natural killer (NK)-resistant monolayer targets is an inhibitory signal to NK cells. In this study, we have analyzed the effect of such effector/target cell interactions on the CD16 (FcRIII) expression on lymphocytes and the role of CD16 and interleukin-2 (IL-2) in the reactivation of their cytolytic machinery. Coculturing peripheral blood mononuclear cells with NK-resistant monolayer cells did not change the percentage of CD16-positive effector cells, although this treatment effectively inhibited their cytotoxicity against NK-sensitive targets. The inhibited effector cells partially regained their activity by incubating for 24 h in medium supplemented with 10% fetal calf serum (FCS), whereas human albumin-, newborn calf serum- or human AB serum-supplemented media had no reactivating effect. Monoclonal class IgG1, IgG2a and IgM anti-CD16 antibodies [Abs; 3G8, CLB-CD16 (CLB-FcR gr1) and Leu 11b], and normal rabbit IgG (NR-IgG) prevented the FCS-mediated reactivation of cytotoxicity, whereas nonreactive control Abs significantly enhanced it. The detection of the CD16 antigen by the monoclonal anti-CD16 Abs Leu 11a and Leu 11c was blocked by the above anti-CD16 Abs and NR-IgG, while the expression of other NK cell-associated surface molecules (CD2, CD56) remained unchanged. Mere blocking of CD16, using a short-term incubation with anti-CD16 Abs, had an insignificant effect on endogenous NK activity, suggesting that CD16 is involved in NK cell (re)activation rather than in the killing process itself In the presence of IL-2, inactivated effector cells also regained their killing activity. The IL-2-induced reactivation was not inhibited by anti-CD16 Abs. The results suggest that FCS-derived factors and soluble nonreactive immunoglobulins enhance the NK activity of down-regulated effector cells via CD16, and that CD16 and IL-2 receptors represent alternative independent pathways of NK cell reactivation.
The effect of short-term exposure (1 month) to a low-pathogen environment (LPE) on NK cell levels in the bone marrow and spleen of immunologically normal CBA/CaJ and B-lymphocyte-deficient CBA/N mice was assessed using both functional (51Cr release) and histological methods. The total NK activity (TNKA), i.e. the percentage specific lysis corrected for changes in whole organ cellularity, of the spleens of LPE-exposed CBA/CaJ mice was not significantly different from that of conventionally reared control mice of that strain (112%), while TNKA of the bone marrow of LPE-exposed mice fell to 27% of that of the bone marrow of conventionally reared controls. TNKA of the spleen and bone marrow of LPE-exposed CBA/N mice was reduced (30%) and elevated (120%), respectively, relative to their conventionally reared counterparts. The numbers of asialo-GM-1-positive (ASGM-1+) lymphoid cells, putative NK cells, in CBA/CaJ spleens were more numerous in conventionally reared than in LPE-exposed mice (4.9 X 10(6) vs. 2.7 X 10(6), and were also more numerous in the bone marrow of conventionally reared mice than in LPE-exposed animals (8.0 X 10(4), vs. 3.0 X 10(4) cells). Similarly, in LPE-exposed CBA/N mice, the numbers of ASGM-1+ lymphoid cells in the spleens and bone marrow were lower (2.7 X 10(6) and 5.4 X 10(4), respectively) than those of the spleens and bone marrow of their conventionally reared counterparts (3.8 X 10(6) and 10.0 X 10(4), respectively). The results demonstrate that short-term maintenance in an LPE affected the NK cells of both the spleen and bone marrow of immunologically normal and B-lymphocyte-deficient mice in a strain-specific manner and suggest that the external environment may regulate NK cell production and turnover.
By employing a distinctive feature of natural killer (NK) cells, i.e., spontaneous target cell binding, the present study aimed to follow a relatively large cohort of target-binding cells (TBC), subdivided according to size and the presence of the radioautographically labelled nucleotide, tritiated thymidine, incorporated during a 1- or 6-hour exposure period. Labelling among all TBC in the spleen was 5% by 1 h after a single injection of the isotope and this value did not change significantly after 6 h of isotope exposure. There was an insignificant increase in the labelling index of spleen-localized, labelled, large TBC throughout and beyond the isotope exposure period for at least 48 h, concomitant with a significant increase in the labelling index of spleen-localized, small TBC during the same period. In the bone marrow, small TBC showed only 2% labelling by 1 h after a single injection of isotope, while 21% of large TBC in that organ were synthesizing DNA during the same period. The results are consistent with a precursor-product relationship between large and small TBC within the bone marrow but not the spleen, and with a bone marrow-to-spleen migration of small (+/- large) TBC. Moreover, a minor population of large TBC was detected in the spleen with kinetic characteristics distinct from those of the bone marrow.
The relationship of the Kurloff cell (KC), guinea pig blood mononuclear cell with natural killer (NK) activity, to a known cell lineage was established. Using indirect immunoperoxidase staining and flow cytometric analysis, numerous monoclonal antibodies directed against guinea pig macrophage antigen, Ia antigen or different T lymphocyte markers and a polyclonal anti-IgM serum were tested in unimmunized estrogenized animals. We excluded any relationship between KC and the monocytic macrophage lineage (MR-1-) and between KC and the B lymphocyte lineage (CT10- and IgM-). The KC immunophenotype was pan T CT7 positive but 8BE6 (mature thymocyte) and CT6 (cytotoxic suppressor T lymphocyte) negative. Since KC displays an NK activity, this cell may be classified among the NK effector cells exhibiting some T lymphocyte markers.
