The two-step leukocyte migration inhibition factor (LIF) assay. Its use in evaluation of cellular immune function in patients with immunodeficiency diseases.

Administration of interferon (IF) inducers to mice enhances the uptake of antibody-coated erythrocytes (EA) by peritoneal macrophages. To evaluate the role of induced IF in macrophage activation, serum IF titers and phagocytosis of EA by macrophages were determined in recombinant inbred (RI) mice inoculated with Newcastle disease virus (NDV). RI strains carry either a “high” or a “low” response allele for a gene that controls their IF titers induced by NDV. C × BH and C × BK strains, both high responders for IF induction, were also found to be high responders for enhancement of phagocytosis by NDV. Conversely, strains C × BD, C × BI and C × BJ, low responders for IF induction, were also shown to be low responders for phagocytosis of EA by macrophages. In contrast, phagocytosis of EA by macrophages from high responder C × BH mice and low responder C × BD mice was similarly enhanced by the administration of a lipopolysaccharide. When data from all NDV-inoculated mice were analysed, a significant correlation was obtained between serum IF titers and the percentage of macrophages that ingested four or more EA. The results are compatible with two main possibilities: (i) IF induced by NDV enhances phagocytosis of EA by macrophages; or (ii) a macrophage-activating factor different from IF is released together with IF in response to NDV and the activity of this factor correlates with serum IF titers.     

Extracellular products of type IIIStreptococcus agalactiae and their relationship to virulence

Various human isolates of type III group B streptococci (GBS) orStreptococcus agalactiae could be divided into two distinct groups (high and low producers) on the basis of their in vitro production of extracellular type-specific antigen (ETSA). The high ETSA producers were shown to be significantly more virulent in mice than were the low producers. In an effort to examine the possibility that purified extracellular products (ETSA, neuraminidase, or protease_ had a significant effect on GBS virulence in the mouse model, mice received either 1.0 ml of organisms intraperitoneally (IP) or 1.0 ml of organisms IP plus 0.1 ml IP of the appropriate purified extracellular products. Only purified ETSA demonstrated a substantial decrease (1.0 log₁₀) in the 50% lethal dose (LD₅₀) values and only for a high ETSA producing strain. Serum from mice infected with a high ETSA producer contained approximately 12.5 μg/ml of the ETSA, whereas serum from mice infected with a low ETSA producer contained no detectable ETSA. These data imply that the two different types of organisms (high and low ETSA producers) have somewhat different mechanisms of pathogenicity in the mouse model.     

Virus replication and high-titered interferon production in human leukocyte cultures inoculated with Newcastle disease virus

Wheelock, Frederick E. (Western Reserve University, Cleveland, Ohio). Virus replication and high-titered interferon production in human leukocyte cultures inoculated with Newcastle disease virus. J. Bacteriol. 92:1415-1421. 1966.-High titers of interferon (20,480 culture-protecting units per ml) are produced in freshly prepared human leukocyte cultures inoculated with a Newcastle disease virus (NDV)-cell multiplicity of 1:1. NDV replicates to low titers in these cultures. Incubation of leukocytes at 37 C for 24 hr prior to inoculation of NDV results in almost complete loss of detectable interferon production, but virus replicates to higher titers than in the freshly prepared cultures. In contrast, no diminution of interferon production in response to phytohemagglutinin (PHA) occurs on 24 hr of incubation of cultures prior to addition of PHA. Experiments with cultures of predominantly pure cell fractions of peripheral blood indicate that the lymphocyte fraction produces interferon in response to either NDV or PHA, and that polymorphonuclear leukocytes produce no interferon in response to these agents. These studies suggest a hitherto unsuspected ability of human lymphocytes to produce high titers of interferon in vivo.     

Inhibition of macrophage migration by virus-induced interferon preparations.

It was found that a preparation of mouse L cell interferon induced by Newcastle disease virus (NDV) possessed not only interferon activity but also inhibitory activity upon migration of guinea pig peritoneal macrophages (MIF activity). These activities were also observed in a preparation of human leukocyte interferon induced by NDV. The interferon and MIF activities shared common characteristics in the dose response, time course of in vitro production, thermal stability, sensitivity to trypsin and periodate, and elution pattern in CM-Sephadex column chromatography. However, gel filtration pattern with Sephadex G-100 showed two separate peaks. Fractions collected from the first peak, corresponding to a molecular weight of about 45 000, had only the MIF activity, while those collected from the second peak, corresponding to a molecular weight of about 30 000, had both the interferon and MIF activities. A preparation of mouse brain interferon induced by Japanese encephalitis virus had a much weaker MIF activity than the L cell interferon, although these preparations were equal in interferon activity (5000 units/ml).     

Interferon Induction in Rabbit Cells Irradiated with UV Light

UV irradiation of a continuous line of rabbit kidney cells (RK13) was used as a tool for the study of the mechanism of interferon induction. Irradiation of cells prior to their exposure to Newcastle disease virus (NDV) resulted in a dose-dependent decrease in interferon production. The inhibition of total cellular RNA synthesis by UV irradiation in uninduced cultures was similar to the inactivation curve of interferon production in NDV-induced cultures. In contrast, the production of interferon with polyinosinate-polycytidylate (poly[I].poly [C]) paradoxically was enhanced in cells irradiated with a wide range of doses of UV. However, in cells stimulated with poly(I).poly(C) and "superinduced" by the sequential addition of cycloheximide and actinomycin D, the rate of inactivation of interferon production by UV light was similar to that observed with NDV. These results are not inconsistent with the idea that both poly(I).poly(C) and NDV stimulate the same interferon gene(s), but indicate that the mechanism controlling its expression may be different for each inducer.     

Production of Mouse Interferon with High Titers in a Large-Scale Suspension Culture System1

L-MS cells, adapted to grow in suspension, were obtained by selection from a high interferon (IF)-producing line of mouse L cell monolayers. A large volume of L-MS cells (20 liters or more; 1–2 × 10¹⁰ cells) was readily grown in a spinner culture, retaining their ability to produce high yields of IF in serum-free medium following induction with Newcastle disease virus (NDV). The optimal condition for the production of IF in the suspension culture of L-MS cells was established. The system also proved itself to be susceptible to IF induction by polyinosinic-polycytidylic acid (Poly I · Poly C) and by NDV inactivated with ultraviolet light (NDV-UV). By employing the present system, large quantities of mouse IF of a high titer could be routinely prepared.     

Viral Ribonucleic Acid Synthesis by Newcastle Disease Virus Mutants Isolated from Persistently Infected L Cells: Effect of Interferon

The synthesis of different viral ribonucleic acid (RNA) species was studied in chick embryo (CE) and mouse L-cell cultures infected with the Herts strain of Newcastle disease virus (NDV(o)) and a mutant isolated from persistently infected L cells (NDV(pi)). In CE cell cultures, both viruses synthesized significant amounts of 54, 36, and 18S RNA. However, in L cells, synthesis of 54S virion RNA was markedly reduced. From these results, it seems likely that the low yield of infective virus in L cells is due to a deficient synthesis of 54S RNA in this host. On this basis, however, it is apparent that the "covert" replication of NDV(o) in L cells is due to factors other than viral RNA synthesis. When low concentrations of interferon were used to pretreat CE cells, a differential effect on the synthesis of various RNA species was observed. The 18S RNA of NDV(o) was more sensitive to interferon action than the 36 and the 54S RNA species. In contrast, the 18S RNA of NDV(pi) was less sensitive than the 36S and the 54S RNA. The inhibition of 54S RNA synthesis correlated with the reduction of viral yield and explained the greater sensitivity of NDV(pi) to interferon.     

Inhibition of Macrophage Migration by Virus-Induced Interferon Preparations1

It was found that a preparation of mouse L cell interferon induced by Newcastle disease virus (NDV) possessed not only interferon activity but also inhibitory activity upon migration of guinea pig peritoneal macrophages (MIF activity). These activities were also observed in a preparation of human leukocyte interferon induced by NDV. The interferon and MIF activities shared common characteristics in the dose response, time course of in vitro production, thermal stability, sensitivity to trypsin and periodate, and elution pattern in CM-Sephadex column chromatography. However, gel filtration pattern with Sephadex G-100 showed two separate peaks. Fractions collected from the first peak, corresponding to a molecular weight of about 45 000, had only the MIF activity, while those collected from the second peak, corresponding to a molecular weight of about 30 000, had both the interferon and MIF activities. A preparation of mouse brain interferon induced by Japanese encephalitis virus had a much weaker MIF activity than the L cell interferon, although these preparations were equal in interferon activity (5000 units/ml).     

Induction of xanthine oxidase and depression of cytochrome P-450 by interferon inducers: genetic difference in the responses of mice

Interferon and interferon inducing agents depress hepatic cytochrome P-450 systems. They also induce hepatic xanthine oxidase activity. It has been suggested that free radicals produced by xanthine oxidase may cause the loss of P-450. High titers of serum interferon are induced by poly IC (poly riboinosinic acid.polyribocytidylic acid) in both C57Bl/6J and C3H/HeJ mice; Newcastle disease virus (NDV) induces a high titer of interferon in C57Bl/6J mice but not in C3H/HeJ mice. The induction of xanthine oxidase activity by NDV in C3H/HeJ mice was less than half that seen in C57Bl/6J mice, thus demonstrating a relationship between the induction of xanthine oxidase, the depression of P-450 and a genetically determined difference in responsiveness of mice to interferon inducers.     

Mechanism for the suppression of gamma-interferon responsiveness in mice after thermal injury

As reported previously, gamma-interferon production was decreased after the administration of inducers to thermally injured mice as compared with noninjured controls. Similarly, spleen cells from injured mice had decreased ability to produce interferon in vitro after stimulation with inducers. The present study demonstrated that the decrease in interferon production was associated with the presence of suppressor cells in the spleen of burned mice that were capable of inhibiting interferon production by normal splenic lymphocytes in vitro. Passive transfer of spleen cells containing suppressor cell activity derived from injured mice induced suppression in normal mice, and the time of the appearance of suppressor cell activity in injured mouse spleens closely approximated the time of the appearance of the suppression of interferon production observed in mice after thermal injury. The suppressor cells were characterized as a population of macrophages by the following: they adhered to plastic surface and could be removed from spleen cells by carbonyl-iron treatment; treatment of plastic-adherent cells with anti-Thy-1.2 and anti-mouse immunoglobulin antisera followed by complement failed to abrogate the suppression produced by these cells.     

Interferon structural genes do not participate in quantitative regulation of interferon production by If loci as shown in C57BL/6 mice that are congenic with BALB/c mice at the alpha interferon gene cluster.

Previous studies have shown that serum interferon (IFN) production in mice is quantitatively influenced by If loci, whose alleles determine high or low production. Although different loci influence IFN production in response to different inducers, such as Newcastle disease virus, Sendai virus, herpes simplex virus type 1, and polyriboinosinic-polyribocytidylic acid, BALB/c mice are in every instance low producers. It was therefore possible that, in addition to If loci, some feature of the BALB/c structural IFN genes contributed to low production. This was examined in the present work, in which IFN production was measured in two strains of C57BL/6 mice congenic with BALB/c at the murine alpha IFN (IFN-alpha) gene cluster on chromosome 4. One line, HW13 (B6.C-H-15c-H-16c-H-20c-H-21c/By) has a BALB/c fragment on chromosome 4 of at least 35 centimorgans which includes the BALB/c IFN-alpha gene cluster and four loci of the brown histocompatibility complex; the other line, HW13J (B6.C-H-15c/By), has a much shorter fragment (about 15 centimorgans), but it also comprises the BALB/c IFN-alpha gene cluster. We show that these mice, carrying the BALB/c IFN-alpha structural genes on a C57BL/6 background, are high IFN producers when stimulated by Newcastle disease virus, Sendai virus, herpes simplex virus type 1, or polyriboinosinic-polyribocytidylic acid. Thus, the low IFN production of BALB/c mice is not directly due to some feature of the IFN-alpha structural genes but is mainly the result of different alleles at If loci.     

Effect of Antilymphocytic Serum on Circulating Interferon in Mice as a Function of the Inducer

MOST investigators concerned with interferon synthesis in vivo have used the experimental procedure described by Baron and Buckler¹, in which circulating interferon is induced by intravenous administration of viruses. When interpreting results, however, it is difficult to know which cells are responsible for circulating interferon synthesis in the animal. Using a radiobiological approach, we have shown that after an intravenous injection of virus, interferon released into the blood stream of mice originates in cell populations of varying radiosensitivities, depending on the virus inoculated². Myxo-virus-induced circulating interferon production is characterized by high radiosensitivity, for serum interferon titres are decreased by more than 90% in C3H/He mice after one total body X-irradiation of 250 r. Moreover, the species specificity of interferon has enabled us to show that circulating interferon induced by Newcastle disease virus (NDV) is of donor type in xenogeneic radiochimaeras, from which we concluded that cells responsible for interferon synthesis with this virus originate from haemopoietic stem cells^3,4. Both granulocytes and lymphocytes fulfil the criteria of very radiosensitive elements derived from haemopoietic stem cells^5,6. We wish to report that myxovirus-induced circulating interferon production is selectively depressed after administration of antilymphocyte serum (ALS).     

Alterations of interferon production in a mouse model of thermal injury

The effect of thermal injury on the response of interferon (IFN) production in vivo and in vitro after stimulation with eight representative inducers was investigated in a mouse model. The response of mice to immune IFN (IFN-gamma) inducers, staphylococcal enterotoxin A, concanavalin A, and a specific antigen for BCG-sensitized lymphocytes (purified protein derivative) was impaired after a 30% total body surface area third-degree burn. Suppression of IFN-gamma production was observed at day 2 and persisted until day 7 after burn. Decreased IFN-gamma production correlated closely with the percentage of total body surface area burned. When virus type IFN (IFN-alpha/beta) inducers, Newcastle disease virus, polyriboinosinic-polyribocytidylic acid, 10-carboxymethyl-9-acridanone, and E. coli endotoxin, were administered to mice, no change in IFN response was observed after thermal injury. Similar results were obtained when spleen cells obtained from thermally injured mice were stimulated with IFN-gamma inducers in vitro. These studies suggest that although the capacity for IFN-alpha/beta production remains intact in thermally injured mice, IFN-gamma production may be selectively decreased in burned animals and in their spleen cells.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. III. Interferon and cytotoxin induced by the specific antigen as compared with those induced by bacterial lipopolysaccharide

The time course of development and decline of the ability of BCG-infected mice to produce interferon in the serum in response to the intravenous infection of purified protein derivative of tuberculin (PPD) was very similar to that of their systemic hypersensitivity to PPD. A cytotoxic factor (cytotoxin) was produced in parallel with interferon in the serum of BCG-infected mice after stimulation with PPD. The duration of the period in which cytotoxin-production responsiveness to PPD was definitely detectable was much shorter than that for interferon-production responsiveness although the periods for the maximum production of interferon and cytotoxin coincided. The kinetics of release of interferon in the serum of BCG-infected mice after stimulation with PPD did not parallel that of release of cytotoxin. The four kinds of activities, interferons and cytotoxins induced by PPD and lipopolysaccharide (LPS) in the serum of BCG-infected mice, were compared for their stability to heating at 56 C and to treatment at pH 2. The kinetics of inactivation of these four activities differed significantly, when the serum was either heated at 56 C or treated at pH 2. Interferon produced in response to LPS could be neutralized by anti-L cell(NDV) interferon rabbit serum as easily as L cell (NDV) interferon, 16 times as much antiserum was required to neutralize the same amount of interferon in response to PPD, but cytotoxins induced by PPD and LPS were not neutralized at all by the antiserum. From these findings it is thought likely that interferons and cytotoxins induced by PPD and LPS in the serum of BCG-infected mice are different substances, although the antigenic relationship between cytotoxins induced by PPD and LPS remains unknown.     

Factors Affecting the Sensitivity of Different Viruses to Interferon

When the sensitivities to interferon of Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV) were compared by the plaque reduction method in chick embryo cell cultures, NDV was found to be 45-fold more resistant than VSV. This difference was exaggerated when a multiple-cycle yield inhibition method was employed. In marked contrast, when the same viruses were tested by a single-cycle yield inhibition method, the difference in sensitivity to interferon of the two viruses was virtually eliminated. Further investigation showed that, in chick embryo cells exposed to interferon, the resistance to NDV decayed more rapidly than resistance to VSV. This finding explained the divergent results obtained with the two viruses when single- or multiple-cycle replication techniques were employed. Experiments carried out with L cells showed that cellular antiviral resistance decayed much more slowly in these cells than in chick embryo cells. Consequently, when measured by the plaque reduction method in L cells, no difference was observed in the sensitivity to interferon of VSV and NDV(pi), a mutant of NDV which replicates efficiently in L cells. A procedure is suggested for determining the relative sensitivities to interferon of different viruses under conditions which minimize the role of decay of antiviral resistance in the host cells.     

Effect of heat and milk yield on bovine plasma glucocorticoid levels.

This study indicates that at 15 degrees higher producing cattle (milk yield) have higher plasma glucocorticoid concentrations compared to lower producing cattle with glucocorticoid levels appearing to be positively correlated with lactational intensity. Short term thermal (30 degrees) exposure for 18 hr resulted in glucocorticoid levels being markedly lower in high producing cattle compared to low producers. This shift at 30 degrees (after 18 hr) is possibly due to different time sequence of glucocorticoid response to thermal exposure between high and low producing cows. These data support the concept that glucocorticoids assist the animal in efficiently meeting the greater energy demand of lactation and further studies should be undertaken to denote free plasma levels and their utilization by dairy cattle at various levels of milk production.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. I. Enhanced production of interferon and appearance of cytotoxin stimulated by capsular polysaccharide of Klebsiella pneumoniae or bacterial lipopolysaccharide.

Interferon production stimulated by the active substance (neutral fraction) of the capsular polysaccharide of Klebsiella pneumoniae (neutral CPS-K) in BCG-infected mice was compared with that by bacterial lipopolysaccharide (LPS). Prior infection with BCG increased the responsiveness of mice to the lethal effect of neutral CPS-K as well as to that of LPS. Associated with this, BCG-infected mice showed a markedly enhanced ability to produce interferon after stimulation not only by LPS but also by neutral CPS-K. In addition, a cytotoxic factor (cytotoxin) was found to be released in the serum of BCG-infected mice after injection of these inducers. The kinetics of production of interferon and cytotoxin stimulated by neutral CPS-K were very similar to those stimulated by LPS. The time pattern of cytotoxin production was not in parallel with that of interferon production. Interferon reached a peak 2 hr and cytotoxin 3 hr after injection with these inducers. Interferon and cytotoxin produced by neutral CPS-K showed essentially the same stabilities to heating at 56 C and to treatment at pH 2 respectively as those produced by LPS. Interferon was inactivated by heating at 56 C more rapidly than cytotoxin. Cytotoxin was inactivated by treatment at pH 2 for 24 hr, whereas interferon activity was well preserved after this treatment. These results suggest that both activities are the result of different substances.     

The effect of stress on interferon production in mice

In order to study the effect of stress on the interferon (IFN) production, we determined the IFN-alpha/beta and -gamma producing capacities in restraint-stressed and SART-stressed mice. Restraint-stress caused not only a significant reduction in body weight, but also a reduction in the weight of spleen. The IFN-alpha/beta producing capacity by Newcastle disease virus (NDV) was significantly lower in the stressed mice. The IFN-gamma producing capacity by both Con-A and PHA-P was also depressed by this stress. On the other hand, SART-stress, whose severity was thought to be mild because no loss of body and spleen weights was seen, did not affect IFN-alpha/beta and -gamma producing capacities. These results suggest that the suppression of IFN producing capacity is dependent on the severity of physical stress.