Ultrastructure of Klebsiella O3 lipopolysaccharide isolated from culture supernatant: structure of various uniform salt forms

Various uniform salt forms of Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from culture supernatant were prepared as follows. Basic materials present in KO3 LPS were rigorously removed by electrodialysis and the electrodialyzed KO3 LPS was neutralized with NaOH, KOH, NH4OH, Ca(OH)2, tris(hydroxymethyl)aminomethane, or triethylamine. The ultrastructure of the uniform salt forms of KO3 LPS was examined using preparations stained with uranyl acetate. The sodium, potassium, ammonium, and trisaminomethane salt forms were structurally very similar to the natural form of KO3 LPS which consisted of a mixture of flat ribbon-like structures (average width of 16 nm and average thickness of 7 nm) and spheres with various diameters, both covered with fine hairy structures. When KO3 LPS was converted to the triethylamine salt form, the ribbon-like structures were disrupted into very small granules (7-9 nm X 9-15 nm). The calcium salt form consisted of particles and rods of various sizes and ribbon-like structures which were markedly extended (maximum width of 50 nm) and presented irregular shapes. When converted to the calcium salt form, the ribbon-like structures were extended and eventually divided into particles and rods. For reasons still unknown, the sodium salt of KO3 LPS was mostly stained positively with uranyl acetate in contrast to the natural form and the other uniform salt forms which were always negatively stained. In the positively stained preparation of the sodium salt form, it was clearly shown that the ribbon-like structures consisted of a bilayer.     

Correlation between strong adjuvanticity of Klebsiella O3 lipopolysaccharide and its ability to induce lnterleukin-1 secretion

Adjuvant activity of Klebsiella O3 lipopolysaccharide (KO3 LPS) in augmenting antibody response and delayed-type hypersensitivity to protein antigens in SMA mice was much stronger than that of LPS from Escherichia coli O55 and O127 (EO55 LPS and EO127 LPS). Relationship between strength of the adjuvant activity and that of the ability to induce interleukin-1 (IL-1) secretion by peritoneal macrophages from C3H/HeN or SMA mice was investigated using these three kinds of LPS. When supernatant samples of macrophages cultured at 37 °C for 24 hr in the presence of 5 μg/ml LPS were assayed by their mitogenic effect on thymocytes from C3H/HeJ mice, KO3 LPS induced the secretion of about four to six times greater amounts of IL-1 activity than did EO127 LPS. When concentration of LPS used for stimulation of macrophages was varied from 0.1 to 50 μg/ml, KO3 LPS induced the secretion of definitely greater amounts of IL-1 activity than did EO55 LPS and EO127 LPS throughout the LPS concentrations tested. Nearly the same amount of IL-1 activity as that produced by 10 μg/ml EO55 LPS or 50 μg/ml EO127 LPS could be produced by 1.0 μg/ml or lower concentrations of KO3 LPS.     

Ultrastructure of Klebsiella O3 Lipopolysaccharide Isolated from Culture Supernatant: Comparison with Other Lipopolysaccharides

Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from the culture supernatant, which was found to exhibit a very strong adjuvant activity in augmenting antibody response and delayed-type hypersensitivity to protein antigens in mice, was examined by electron microscopy. When negatively stained with uranyl acetate or ammonium molybdate, the KO3 LPS was found to consist principally of flat ribbon-like structures branching freely (average width 16 nm and average thickness 7 nm) and to contain a small proportion of spheres (diameter 20–50 nm), both structures covered with fine hairy structures (average length approximately 10 nm). When the polysaccharide of KO3 LPS was stained by the periodic acid-thiosemicarbazide-silver proteinate procedure, silver granules were deposited on the ribbon-like structures and around the spheres, suggesting that the polysaccharide moiety is located on their surface and that the fine hairy structures consist of the polysaccharide moiety. Comparison by means of preparations stained with uranyl acetate or ammonium molybdate showed that KO3 LPS isolated from the culture supernatant has structural features in common with KO3 LPS isolated from bacterial cells, Escherichia coli O9 LPS isolated from the culture supernatant, and E. coli O127 LPS isolated from bacterial cells. On the basis of the present results, schematic representations of the common physical structure of LPS were drawn; the fine hairy structures attach to the wide surface of the flat ribbon-like structures along their lateral margin.     

Adjuvant activity of Klebsiella O3 lipopolysaccharide: inhibition of the adjuvant activity by concanavalin A

Klebsiella O3 lipopolysaccharide (KO3 LPS) was found to exhibit extraordinarily strong adjuvant activity in augmenting antibody responses and delayed-type hypersensitivity (DTH) to protein antigens in mice. The O-specific polysaccharide chain of KO3 LPS consists of alpha-mannoside. We investigated the effect of concanavalin A (Con A) or succinyl Con A, which is known to bind to alpha-mannoside, on the adjuvant activity of KO3 LPS in augmenting DTH to ovalbumin. When KO3 LPS was mixed with Con A prior to injection, the strong adjuvant activity of KO3 LPS in augmenting DTH was inhibited and the degree of inhibition depended upon the dose of Con A. An equal amount of Con A elicited nearly complete inhibition of the adjuvant activity of KO3 LPS, Con A at 1/10 the amount of LPS elicited partial inhibition, and Con A at 1/100 the amount of LPS showed no inhibition. An equal amount of succinyl Con A, which induced less marked aggregation of KO3 LPS than Con A, elicited inhibition of the adjuvant activity of KO3 LPS to an extent similar to that by Con A. On the other hand, Con A or succinyl Con A bound to KO3 LPS did not impair in any way the lethal toxicity of KO3 LPS for mice which is known to be due to the lipid A moiety. From these findings it is concluded that the strong adjuvant activity of KO3 LPS does not solely depend upon the lipid A moiety but the O-specific polysaccharide moiety plays an important role in expression of the adjuvant activity.     

Potent adjuvant action of lipopolysaccharides possessing the O-specific polysaccharide moieties consisting of mannans in antibody response against protein antigen

It was previously reported that Klebsiella O3 lipopolysaccharide (LPS) exhibits extraordinarily strong adjuvant activity in augmenting antibody response against protein antigens in mice compared with other kinds of LPS, for example, LPS from Escherichia coli O55, O111, and O127 and Salmonella enteritidis. The present study was undertaken to clarify the relationship between the strong adjuvant activity in augmenting antibody response against deaggregated bovine gammaglobulin and the chemical structure of LPS. Among LPS from Klebsiella O1, O4, O5, and O7, only O5 LPS exhibited nearly the same degree of the strong adjuvant activity as did O3 LPS. The adjuvant activity of the other LPS was very weak in a degree similar to that of LPS from E. coli O55 and O127. Even when the natural forms of Klebsiella O3 LPS and O1 LPS were converted to various defined uniform salt forms, their adjuvant activity did not significantly differ from that of the respective natural forms. It is therefore unlikely that the difference in strength of the adjuvant activity between Klebsiella O3 LPS and O1 LPS is due to the difference in their salt forms. The common feature in the structures of Klebsiella O3 LPS and O5 LPS is their O-specific polysaccharide chains consisting of the mannose homopolysaccharides (mannans). LPS from E. coli O8 and O9, the O-specific polysaccharide chains of which consist of the mannans, also exhibited much stronger adjuvant activity than do LPS from E. coli O55 and O127, and the strength of the adjuvant activities of the former two was comparable to that of LPS from Klebsiella O3 and O5. On the other hand, LPS from Klebsiella O3 and O5 and E. coli O8 and O9 showed the ability to activate B lymphocytes polyclonally in vivo in a degree similar to that of the other kinds of LPS. From the present results it can be concluded that LPS possessing the O-specific polysaccharide moieties consisting of the mannans exhibit extraordinarily strong adjuvant activity in augmenting antibody response against protein antigen.     

Adjuvant activity of Klebsiella O3 lipopolysaccharide: no contribution of proteins to the expression of adjuvant activity

Previously we found that Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from culture supernatant of strain Kasuya (O3: K1) or its decapsulated mutant strain LEN-1 (O3: K1-) exhibited very strong adjuvant activity in augmenting antibody response and delayed-type hypersensitivity to protein antigens in mice. The preparation of KO3 LPS after deproteinization by four cycles of treatment with chloroform-butanol (5: 1) usually contained a small percentage of proteins and a definite amount of another antigen which was destroyed by heating at 100 C for 1 hr. This antigen proved to be derived from type 1 fimbriae which are responsible for mannose-sensitive hemagglutination of guinea pig erythrocytes. The preparation of KO3 LPS isolated from culture supernatant of the strains which did not produce type 1 fimbriae exhibited strong adjuvant activity similar to that of the preparation from those which produced them. The preparation of KO3 LPS treated with hot phenol water which is known to remove lipid A-associated proteins exhibited a similar strong adjuvant activity. The preparation of KO3 LPS after extensive deproteinizing, two cycles of pronase treatment followed by ten cycles of treatment with chloroform-butanol, no longer contained detectable amounts of proteins and the fimbrial antigen, but this preparation also exhibited similar strong adjuvant activity. Moreover, there was no difference in strength of the adjuvant activity between the preparation of KO3 LPS isolated from culture supernatant and that isolated by the phenol method from bacterial cells. The present study demonstrates that the strong adjuvant activity of the preparation of KO3 LPS does not depend in any way on proteins contaminating the preparation.     

Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella: relationship between lattice formation and uniform salt forms

Various uniform salt forms of an R-form lipopolysaccharide (LPS) extracted from Klebsiella strain LEN-111 (O3-:K1-) were prepared and their ultrastructure was examined. The LPS, which was extracted by the phenol-water method, freed from contamination with RNA by treatment with RNase, and precipitated by addition of two volumes of 10 mM MgCl2-ethanol, was used as the original preparation for uniform salt forms. The original LPS preparation formed a hexagonal lattice structure with a lattice constant of 14.9 +/- 0.2 nm. The LPS after electrodialysis retained the ability to form a hexagonal lattice structure, although its lattice constant was large (18.7 +/- 0.5 nm) and the lattice structure of the electrodialyzed LPS was labile at pH 8.0 in contrast to that of the original LPS preparation. The magnesium salt form of the LPS formed essentially the same ordered hexagonal lattice structure (lattice constant of 15.0 +/- 0.2 nm) as that of the original LPS preparation. The calcium and ammonium salt forms formed a hexagonal lattice structure, but the lattice constants of the calcium and ammonium salt forms were larger (18.6 +/- 0.6 nm and 19.3 +/- 0.4 nm, respectively) than that of the magnesium salt form. The sodium and potassium salt forms consisted of freely branching ribbon-like structures with an average width of 13 nm and an average thickness of 9 nm. The triethylamine salt form consisted principally of short rods (10 nm X 9-13 nm).     

Production of murine collagen-induced arthritis using Klebsiella pneumoniae O3 lipopolysaccharide as a potent immunological adjuvant.

Collagen-induced arthritis (CIA) was produced in mice with non H-2q and H-2r haplotypes by repeated immunization of porcine type-II collagen (CII) together with Klebsiella O3 lipopolysaccharide (KO3 LPS) as an immunological adjuvant. Histological changes that appeared in joints of repeatedly immunized mice were characterized by destruction of normal joint structure, synovial hyperplasia with proliferation of synovial cells, and infiltration of inflammatory cells. No such lesions were produced in mice receiving repeated injections of CII alone or KO3 LPS alone. Development of the humoral antibody and the delayed-type hypersensitivity to CII was exclusively found in mice immunized with the mixture of CII and KO3 LPS. It was therefore suggested that arthritis lesions induced by repeated immunization with the mixture of CII and KO3 LPS might be caused by an autoimmune mechanism, and that the experimental model might be useful for characterization of human rheumatoid arthritis (RA).     

Experimental murine model for autoimmune enterocolitis using Klebsiella pneumoniae O3 lipopolysaccharide as a potent immunological adjuvant

An experimental model for autoimmune enterocolitis was produced in mice by repeated immunization of homologous colon extract together with Klebsiella 03 lipopolysaccharide (KO3 LPS) as an immunological adjuvant. Histological changes in the intestinal lesions were characterized by infiltration with polymorphonuclear leukocytes in the lamina propria, muscularis mucosae and submucosa of repeatedly immunized mice. No such intestinal lesions were produced in mice receiving injections of colon extract alone or KO3 LPS alone. Development of the autoantibody and delayed-type hypersensitivity against colon extract were found in mice immunized with the mixture of colon extract and KO3 LPS. Distinct positive staining was detected specifically on the columnar epithelium of villi. Sera from hyperimmunized mice defined organ-specific antigens present in the intestine. Therefore, it was suggested that the intestinal lesions might be caused by an autoimmune mechanism.     

A possible correlation between histological changes in regional subcutaneous tissue induced by bacterial lipopolysaccharides and their adjuvant activities

Previously it was demonstrated that Klebsiella pneumoniae O3 lipopolysaccharide (KO3 LPS) exhibited much stronger adjuvant action on antibody response to subcutaneously (s.c.) injected sheep red blood cells or deaggregated bovine serum albumin than did other kinds of LPS, the R-form LPS lacking the O-specific polysaccharide chain of KO3 LPS (R-LPS), and the lipid A fractionated from KO3 LPS. We compared histological changes in the regional subcutaneous tissues of mice injected subcutaneously (s.c.) with KO3 LPS, the lipid A, and R-LPS. At the early stage after injection, KO3 LPS induced the infiltration of a large number of inflammatory cells, mainly polymorphonuclear leukocytes (PMN), at the site of injection. Neither R-LPS nor the lipid A induced the accumulation of PMN so much as KO3 LPS did. When injected s.c. with LPS from Escherichia coli O111 (EO111 LPS) and O55 (EO55 LPS), and Salmonella enteritidis (Sent LPS), the appearance of PMN at the regional site was much less than KO3 LPS. KO3 LPS could accumulate more 51Cr-labeled leukocytes at the injection site than EO111 LPS and Sent LPS. Administration of acetylsalicylic acid, which can inhibit leukocyte migration in inflammatory lesions, suppressed its adjuvant action. It was therefore suggested that the strong adjuvant action of KO3 LPS in s.c. injection might be dependent on its potent capability of accumulating PMN at the regional subcutaneous tissue. Furthermore, at the late stage after injection, the formation of several lymphoid follicles at the regional site was seen only in mice injected with KO3 LPS. It might be also related to the strong adjuvant action of KO3 LPS.     

Divalent cations affect chain mobility and aggregate structure of lipopolysaccharide from Salmonella minnesota reflected in a decrease of its biological activity

The physicochemical properties and biological activities of rough mutant lipopolysaccharides Re (LPS Re) as preformed divalent cation (Mg²⁺, Ca²⁺, Ba²⁺) salt form or as natural or triethylamine (Ten⁺)-salt form under the influence of externally added divalent cations were investigated using complementary methods: Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopic (FT-IR) measurements for the β ↔ α gel to liquid crystalline phase behaviour of the acyl chains of LPS, synchrotron radiation X-ray diffraction studies for their aggregate structures, electron density calculations of the LPS bilayer systems, and LPS-induced cytokine (interleukin-6) production in human mononuclear cells. The divalent cation salt forms of LPS exhibit considerable changes in physicochemical parameters such as acyl chain mobility and aggregate structures as compared to the natural or monovalent cation salt forms. Concomitantly, the biological activity was much lower in particular for the Ca²⁺- and Ba²⁺-salt forms. This decrease in activity results mainly from the conversion of the unilamellar/cubic aggregate structure of LPS into a multilamellar one. The reduced activity also clearly correlates with the higher order - lower mobility - of the lipid A acyl chains. Both effects can be understood by an impediment of the interactions of LPS with binding proteins such as lipopolysaccharide-binding protein (LBP) and CD14 due to the action of the divalent cations.     

Strong interaction of lipopolysaccharides possessing the mannose homopolysaccharides with complement and its relation to adjuvant action

LPS from Klebsiella pneumoniae O3 (KO3 LPS) exhibited an extremely high anticomplementary activity by the hemolysis assay using human sera. The free lipid A isolated from KO3 LPS by acid hydrolysis and R form LPS from a mutant lacking the O-specific polysaccharide portion possessed lower anticomplementary activity, and the O-specific polysaccharide fraction isolated from KO3 LPS alone did not activate the C system. It was suggested that the O-specific polysaccharide moiety enhanced the C activation by the lipid A portion. This was also supported by the finding that modification of the O-specific polysaccharide moiety with Con A or tyramine decreased anticomplementary activity of KO3 LPS, and that the other LPS preparations possessing the mannose homopolysaccharides as the O-specific polysaccharide portions such as KO3 LPS, such as LPS from Klebsiella O5, Escherichia coli O8 and O9, exhibited a high anticomplementary activity. KO3 LPS could activate the C system in either the classical or the alternative pathway, whereas the lipid A or R form LPS activated the classical pathway alone. The intensity of anticomplementary activity of LPS was parallel to that of their adjuvant action on antibody response to deaggregated BSA. The role of the anticomplementary activity in the expression of the adjuvant action of LPS is discussed.     

The role of the structural parts of bacterial lipopolysaccharide in its direct immunosuppressive activity]

The direct immunosuppressive activity of lipopolysaccharides (LPS) and their structural parts (O-chains, R-core, lipid A), obtained from Salmonella, Pseudomonas and Burkholderia, was studied. LPS preparations were extracted by the phenol-water method. Structural parts of LPS were obtained by acetic acid hydrolysis and gel filtration. The study demonstrated that all these preparations, when injected intraperitoneally into mice, did not affect the level of delayed-type hypersensitivity (DTH) to the test antigen in the animals. After redox treatment all LPS preparations became capable of suppressing DTH. After redox treatment such immunosuppressive activity could be observed in lipid A, while O-specific chains and R-core remained inactive. After phenol treatment immunosuppressive activity disappeared. Chemical groups capable of activation were likely to be located in lipid A or in lipid A-associated protein.     

Delayed-type hypersensitivity to allogeneic mouse epidermal cell antigens

Footpad swelling response was used to measure the alloantigenicity of epidermal cells (ECs) in delayed-type hypersensitivity (DTH). Strong footpad swelling was oberserved 3 h after the challenge, and it continued for 48 h after the challenge. Genetical incompatibility between the recipients and the ECs was required to induce significant footpad swelling. H-2 or non-H-2 incompatibility between mice and ECs in the sensitization phase sufficed to develop significant footpad swelling. Incompatibility caused by point mutation in the A region induced strong responses when B6. C-H-2 ^bm12 mice were immunized with B6/J ECs, but the disparity in immuno-globulin h (Igh) allotype genes was insufficient. H -Y antigen on ECs could also elicit the DTH response. Semiallogeneic F₁-derived ECs sensitized the parental recipients. The responses were successfully transferred by immune lymph node cells, but not by immune sera. Treatment of these immune lymph node cells with monoclonal antibodies plus complement revealed that the cells responsible for DTH transfer were Lyt-1⁺2^–, Ia^– T cells.Abbreviations used in this paper DNFB 2,4-dinitro-1-fluorobenzene - DTH delayed-type hypersensitivity - ECs epidermal cells - HBSS Hanks' balanced salt solution - MHC major histocompatibility complex - PBS phosphate-buffered saline     

NK T cell activation promotes Chlamydia trachomatis infection in vivo

We used two approaches to examine the role of NK T cells (NKT) in an intracellular bacterial (Chlamydia trachomatis mouse pneumonitis (C. muridarum)) infection. One is to use CD1 gene knockout (KO) mice, which lack NKT, and the other is to activate NKT using alpha-galactosylceramide (alpha-GalCer), a natural ligand of these cells. The data showed a promoting effect of NKT activation on Chlamydia lung infection. Specifically, CD1 KO mice exhibited significantly lower levels of body weight loss, less severe pathological change and lower chlamydial in vivo growth than wild-type mice. Immunological analysis showed that CD1 KO mice exhibited significantly lower C. muridarum-specific IL-4 and serum IgE Ab responses as well as more pronounced delayed-type hypersensitivity response compared with wild-type controls. In line with the finding in KO mice, the in vivo stimulation of NKT using alpha-GalCer enhanced chlamydial growth in vivo, which were correlated with reduced delayed-type hypersensitivity response and increased C. muridarum-driven IL-4/IgE production. Moreover, neutralization of IL-4 activity in the alpha-GalCer-treated BALB/c mice significantly reduced the promoting effect of alpha-GalCer treatment on chlamydial growth in vivo. These data provide in vivo evidence for the involvement of NKT in a bacterial pathogenesis and its role in promoting Th2 responses during infection.     

Murine T cell clones specific for Hymenolepis nana: generation and functional analysis in vivo and in vitro.

To examine the role of the T cell in protective immunity to Hymenolepis nana, H. nana-specific clonal lymphocytes were generated from mesenteric lymph nodes of BALB/c mice infected with H. nana, and some of their functions were analyzed in vitro and in vivo. Following limiting dilution techniques, five clones were generated from mesenteric lymph node cell populations. All of these clones expressed the L3T4⁺, Lyt-2.2⁻ phenotype and proliferated in vitro in response to soluble egg antigen of H. nana. Of five clones, three secreted interleukin 2 (IL-2) and interferon-γ (IFN-γ) after stimulation with egg antigen. Furthermore, these three clones conferred local delayed-type hypersensitivity to egg antigen. The remaining two clones produced interleukin 4 (IL-4) in response to egg antigen, and could not mediate local delayed-type hypersensitivity. Adoptive transfer experiments using clonal lymphocytes were also undertaken in an attempt to define cell types involved in protective immunity. Clonal lymphocytes secreting both IL-2 and IFN-γ transferred protective immunity, equivalent to that obtained by non-cultured-sensitized mesenteric lymph node cells. They were effective in very small numbers. However, clonal lymphocytes that secreted IL-4 did not transfer protective immunity. These results suggest that helper T lymphocytes, especially the Th1 subtype, are involved in protective immunity against H. nana.     

Defective dendritic cell migration and activation of adaptive immunity in PI3Kgamma-deficient mice

Gene-targeted mice were used to evaluate the role of the gamma isoform of phosphoinositide 3-kinase (PI3Kgamma) in dendritic cell (DC) migration and induction of specific T-cell-mediated immune responses. DC obtained from PI3Kgamma-/- mice showed a reduced ability to respond to chemokines in vitro and ex vivo and to travel to draining lymph nodes under inflammatory conditions. PI3Kgamma-/- mice had a selective defect in the number of skin Langerhans cells and in lymph node CD8alpha- DC. Furthermore, PI3Kgamma-/- mice showed a defective capacity to mount contact hypersensitivity and delayed-type hypersensitivity reactions. This defect was directly related to the reduced ability of antigen-loaded DC to migrate from the periphery to draining lymph nodes. Thus, PI3Kgamma plays a nonredundant role in DC trafficking and in the activation of specific immunity. Therefore, PI3Kgamma may be considered a new target to control exaggerated immune reactions.     

Pseudomonas aeruginosa bacteriophage phi PLS27-lipopolysaccharide interactions

We investigated the phi PLS27 receptor in Pseudomonas aeruginosa strain PAO lipopolysaccharide (LPS) by analyzing a resistant mutant. This mutant, which was designated AK1282, had the most defective LPS yet reported for a P. aeruginosa rough mutant; this LPS contained only lipid A, 2-keto-3-deoxyoctonate, heptose, and alanine as major components. In addition, this LPS lacked galactosamine, which is present in the inner core of the LPS of other rough mutants. The loss of galactosamine but only a small decrease in the alanine content indicated that the core of strain PAO LPS differed from the core structure which has been suggested for the LPS of other well-characterized P. aeruginosa strains. Our analysis also indicated that galactosamine residues may be crucial for phi PLS27 receptor activity of the LPS. Electrodialysis of LPS and conversion to salt forms (sodium or triethylamine) influenced the phage-inactivating capacity of the LPS, as did the medium in which the inactivation occurred; experiments performed in 1/10-strength broth resulted in much lower PhI50 (concentration of LPS causing a 50% decrease in the titer of phage during 1 h of incubation at 37 degrees C) values than experiments performed in regular-strength broth. Sonication of the LPS also increased the phage-inactivating capacities of the LPS preparations.     

Antigen-specific augmentation of delayed-type hypersensitivity by immune serum factor in mice: augmentation of anti-tumor cytostatic activity

A humoral factor capable of augmenting delayed-type hypersensitivity antigen specificity (DAF) is present in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DFR). In the present study, a similar factor was identified when xenogeneic tumor cells were used as antigens. This factor also could augment the in vitro anti-tumor cytostatic activity against homologous tumor cells, which correlated with in vivo DFR to the same tumor cells. The cytostatic activity augmented by the transfer of this factor had the following characteristics: The activity appeared in the whole peritoneal exudate cells (PEC) from serum recipients at 4 days after the antigenic challenge. Such an activity was revealed in the collaboration of plastic dish-nonadherent and -adherent PEC as the primary and final effectors, respectively. The appearance of primary effector cells for such an activity was also accelerated in spleen and lymph node cells. However, a sufficient number of macrophages were always required as the final effectors in their functional expression. These primary effectors were sensitized T lymphocytes which produced lymphokine(s) such as macrophage-activating factor(s) and which contributed to this augmented cytostatic activity through the activation of macrophages. Thus, this immune serum factor seems to exert functional expression by accelerating the generation of lymphokine-producing delayed-type T lymphocytes, which is also responsible for cytostatic anti-tumor immunity.     

Effects of six different cytokines on lymphocyte adherence to microvascular endothelium and in vivo lymphocyte migration in the rat

The first step in the migration of lymphocytes out of the blood is adherence of lymphocytes to endothelial cells (EC) in the postcapillary venule. It is thought that in inflammatory reactions cytokines activate the endothelium to promote lymphocyte adherence and migration into the inflammatory site. Injection of IFN-gamma, IFN-alpha/beta, and TNF-alpha into the skin of rats stimulated the migration of small peritoneal exudate lymphocytes (sPEL) into the injection site, and these cytokines mediated lymphocyte recruitment to delayed-type hypersensitivity, sites of virus injection, and in part to LPS. The effect of cytokines on lymphocyte adherence to rat microvascular EC was examined. IFN-gamma, IFN-alpha/beta, IL-1, TNF-alpha, and TNF-beta increased the binding of small peritoneal exudate lymphocyte (sPEL) to EC. IFN-gamma was more effective and stimulated adherence at much lower concentrations than the other cytokines. IL-2 did not increase lymphocyte adherence. LPS strongly stimulated lymphocyte binding. Treatment of EC, but not sPEL, enhanced adhesion, and 24 h of treatment with IFN-gamma and IL-1 induced near maximal adhesion. Lymph node lymphocytes, which migrate poorly to inflammatory sites, adhered poorly to unstimulated and stimulated EC, whereas sPEL demonstrated significant spontaneous adhesion which was markedly increased by IFN-gamma, IL-1, and LPS. Spleen lymphocytes showed an intermediate pattern of adherence. Combinations of IFN-gamma and TNF-alpha were additive in stimulating sPEL-EC adhesion. Depletion of sPEL and spleen T cells by adherence to IFN-gamma stimulated EC decreased the in vivo migration of the lymphocytes to skin sites injected with IFN-gamma, IFN-alpha/beta, TNF-alpha, poly I:C, LPS, and to delayed-type hypersensitivity reactions by 50%, and significantly increased the migration of these cells to normal lymph nodes, as compared to unfractionated lymphocytes. Thus the cytokines and lymphocytes involved in migration to cutaneous inflammation in the rat stimulate lymphocyte adhesion to rat EC in vitro, and IFN-gamma stimulated EC appear to promote the selective adhesion of inflammatory site-seeking lymphocytes.