Václav Větvička

Critical role of Kupffer cell CR3 (CD11b/CD18) in the clearance of IgM-opsonized erythrocytes or soluble β-glucan

Liver macrophages (Kupffer cells) play a major role in blood clearance of both C3-opsonized immune complexes and therapeutic beta-glucan polysaccharides. Human Kupffer cells express three types of C3-receptors: CR1 (C3b-receptor; CD35), CR3 (iC3b- and beta-glucan-receptor), and CR4 (iC3b-receptor; CD11c/CD18). Studies of isolated macrophages have suggested that CR3 is the major receptor mediating capture of either C3-opsonized erythrocytes (E) or beta-glucans. In this investigation, the organ distribution and function of CR3 in the clearance of IgM-opsonized E and soluble CR3-binding polysaccharides were explored in normal vs. CR3-knockout (CR3-KO) mice. Analysis of intravenously (i.v.) injected 125I-anti-CR3 showed that the major vascular reservoir of CR3 was the liver, followed by spleen and lungs. By contrast, clearance of 125I-anti-CR1 appeared to be mediated predominantly by splenic B lymphocytes, as only subsets of splenic macrophages or Kupffer cells were found to express CR1. Clearance of IgM-opsonized 51Cr-E occurred rapidly to the livers of normal mice but was nearly absent in CR3-KO mice. Soluble 125I-beta-glucan exhibited rapid clearance to the liver in normal mice, whereas clearance in CR3-KO mice was significantly reduced. In conclusion, Kupffer cell CR3 plays a crucial role in the clearance of both IgM-opsonized E and beta-glucans.

Effect of human procathepsin D on proliferation of human cell lines

We have used human procathepsin D isolated from supernatant of human breast cancer cell line ZR-75-1 to test its mitogenic activity for a broad spectrum of human-derived cell lines. These cell lines included: breast cancer cell lines ZR-75-1, MDA-MB-436, MBA-MD-483 and MDA-MB-231, B lymphoblastoid cell line Raji, the monocytoid cell line U937, T lymphoblastoid cell line 8402, epitheloid carcinoma cell line HELA, hepatocellular carcinoma cell line Hep G2, breast milk epithelial cell line HBL-100 and angiosarcoma cell line HAEND-1. We have tested the level of proliferation of these cell lines depending on the presence of procathepsin D in the medium. In parallel we have also measured the effect of insulin-like growth factor II under the same experimental conditions. We have found a significant difference between the influence of IGF II and that of procathepsin D. While IGF II promoted in practically the same way the proliferation of all cell lines tested, procathepsin D had a very pronounced effect on breast cancer cell lines only. This finding might help to explain some contradictory results of prognostic significance of procathepsin D in human breast cancer.

CR3 (CD11b/CD18) expressed by cytotoxic T cells and natural killer cells is upregulated in a manner similar to neutrophil CR3 following stimulation with various activating agents

CR3 (CD11b/CD18) functions both as an iC3b-receptor and as an adhesion molecule for cellular ligands such as ICAM-1. Although CR3 has been well characterized on phagocytic cells, much less is known about CR3 on lymphocytes. In this study, the expression of CR3 was examined on resting and stimulated B, T, and natural killer (NK) cells by three-color flow cytometry. Biotinylated anti-CR3 mAb and streptavidin-FITC were used in combination with anti-CD3 mAb conjugated with peridinin chlorophyll-a protein (PerCP) and phycoerythrinlabeled mAbs to CD4, CD8, CD19, or CD56. Among resting lymphocytes, CR3 was expressed on nearly all NK cells (CD56+CD3−), 1% of CD4+CD3+ helper T cells, 7% of CD8+CD3+ cytotoxic T cells, and 20% of B cells (CD19+). Among the 5% of T cells (CD3+) expressing CR3, the majority was CD56+. Incubation of PBMC for 30 min with PMA induced a three- to fivefold increase in CR3 expression on NK cells and a twofold increase on T cells but did not change the expression of CR3 on B cells. This effect of PMA was not blocked by the presence of cycloheximide, suggesting the presence of cytoplasmic (granule) stores of CR3 in these lymphoid cells resembling those previously reported in neutrophils and monocytes. When PBMC were incubated with rIFN-α, rIL-2, β-glucan, or high concentrations of LPS, expression of CR3 on NK cells increased significantly, but ≥4 hr of stimulation was required. Other cytokines (rIFN-γ, rIL-1, rIL-4, rIL-6, TNF-α) and rC5a had no significant effect on CR3 expression. Among NK cells, both the CD56bright and the CD56dim cells expressed CR3, and the expression of CR3 on both of these NK cell subsets was increased in a similar manner by PMA. However, rIL-2 stimulated a greater increase in CR3 expression on CD56bright cells than on CD56dim cells. These studies suggest that CR3 expressed by NK cells or cytotoxic T cells resembles phagocyte CR3 in that cellular activation stimulates increased surface expression of CR3 derived from cytoplasmic reserves of the receptor.

New oligo-β-(1,3)-glucan derivatives as immunostimulating agents

Oligo-beta-(1,3)-glucans were chemically modified in order to introduce a structural variation specifically on the reducing end of the oligomers. The impact of well defined structural modulations was further studied on cancer cells and murin models to evaluate their cytotoxicity and immunostimulating potential.

Effect of cytokines on 5-fluorouracil-mediated immunosuppression

The effect of cytokines on 5-fluorouracil-mediated suppression of antibody response to arsanilic acid-bovine gammaglobulin (ARS-BGG) in high- and low-responding strains of mice was determined. Single i.v. injections of 5-fluorouracil strongly inhibited both primary and secondary antibody responses. The high-responding strain was found to be more sensitive to 5-fluorouracil. Restoration of suppressed IgG antibody responses to ARS-BGG was achieved in vitro by addition of either rIL-4, or rIFN gamma to the culture medium. Similarly, IgA titers recovered following the addition of exogenous rIL-5. The inhibition of IgM production, however, was not influenced by any of the cytokines tested. No significant differences were observed between experimental groups injected with antigen before or after 5-fluorouracil application. Our results suggest that the immunosuppression caused by 5-fluorouracil treatment can be abrogated by the addition of cytokines.

The potentiation of in vitro antibody response in low-responding mice by addition of cytokines.

The effect of exogenous cytokines on the antibody response to ARS-BGG after in vitro immunization in high- and low-responding strains of mice was determined. Twenty units/ml was established to be the optimal dose of cytokines. The low IgG antibody response to ARS-BGG in low-responding B10 mice was increased significantly by addition of either IL-4 or IFN-gamma. Similarly, the lower production of IgA antibodies was elevated by addition of either IL-2, IL-4, IL-5 or IFN-gamma. The levels of IgM were not influenced by any of the cytokines tested. Cytokines, therefore, play a fundamental role in regulating the responsiveness to protein antigen.

Evolutionary paradox of immunity

Dear Editor, Immunity is the sophisticated ability of all multicellular organisms to maintain integrity of its composition and a homeostatic balance of their internal milieu. Immunity determines the genetic stability of each individual, and subsequently the long-lasting evolutionary stability of the species. In reality, immunity keeps all changes induced by internal mutations or by activities and products of enteropathogensat minimum, as the actions possess a capacity to threaten the integrity of an organism. There are two fundamental types of evolution of life on the Earth. At the emergence of first signs of the life, perhaps 3,800 million years ago, the living forms comprised only prokaryotic, microscopic and asexual organisms. In this RNA/DNA world, the evolutionary innovations were originated by gene changes at the nucleotide level (non-lethal mutations, gene duplications, inversion or other genetic manipulations) and also by mutual gene lateral (horizontal) exchange. This Precambrian biota living 85% of Earth Life time was characterized by hypobradytelic evolution (stasis). Emergence of primitive unicellular (colonial) eukaryotes, more than 1,000 million years ago later, was caused by genetic information transfer across species boundaries. This lateral gene transfer resulted in the burst of multicellularity in Edicarian and particularly in Cambrian, some 600 million years ago. This megascopic, aerobic and sexual Phanerozoic biota occupies only 15% of Earth Life time. It is characterized by horotelic evolution with distinguishing extinctions/radiations periods.[1] From evolutionary view, the mobile DNA/RNA elements (transpozons) of viral, prokaryotic or eukaryotic origin are capable overcoming the host immune barriers. It is important to remember that these barriers could represent potential vectors or direct donors of genes bearing new genetic information needed for evolutionary innovations. So why defend against alien, why immunity is needed? It is clear that multicellular organisms have never evolved alone in a splendid isolation from other living beings.[2] Not evolution, but co-evolution formatted life since its dawn on the Earth. Co-evolution has been accomplished by means of horizontal information transition, which seems to be a major evolutionary force. Consequently, living organisms as thermodynamically open systems inevitably must exchange, accept, and transform information. Their existence, cooperation/competition, and adaptive radiation are simultaneously dependent on the defense of their internal integrity. All Phanerozoic eukaryotes are endowed by various forms of defense of “self,” which in fact are constricted by their basic body patterns.[3] On the other hand, genomes are not protected by any immune system. Why did sophisticated immune systems evolve to protect the integrity of an individual, when the genomes are open for entering and accepting “non-self” genes, and what is the evolutionary justification of defense of “self?” We must be aware that actual genomes of all living beings are synologs, the agglomerates of autochthonous genomes with xenologic, alien genetic contributions from others. Organisms that express properties and products from foreign genes are numerous. Even in humans, according International Human Genome Sequencing Consortium, mobile genetic elements form as well as a half of his genome.[4] What happened before the emergence of gnathostomian vertebrates? The groups of virulence genes could transfer new biological information into the genome of placoderms. It could be a benign infectious process that initated the formation of a new defense strategy. It is possible that adaptive immunity developed as a result of some infectious process. What, for decades, appeared as a result of immunological Big Bang most probably developed throughout tens of million of years by slow, step-by-step adoption of the genes necessary for out of genome diversity. V(D)J recombination of the imunoglobulin family became the most successful system of adaptive immunity.[5] It may be hypothesized that the primordial lymphoid cell lineage of these predecessors of gnathostomes was sufficiently plastic for acceptation of recombination-activating genes (RAG) cassettes, which allowed the use of molecules of immunoglobulin super family as defense weapons (antibodies, etc.). The majority of agnathostomes with their adaptive immunity being based on LRR (leucine-rich repeat) proteins disappeared more than 400 million years ago and the two current orders consist of, compared to immunologically more successful vertebrates, only handful of species.[6] Thus the onset of adaptive immune strategy is tightly connected with lateral gene transfer. However, we still know little about the natural pressures leading to the creation of the vertebrate type of immunity, nor about the time at which the first sign of this immunity emerged. It is probable that adaptive immunity evolved against the background of the invertebrate defense reactions which were tending toward some degree of specificity on the phylogenetical level of the first chordate vertebrate ancestors.[3] So the question is: Do we need immunity? It might be possible to conclude that immune systems occurred more by pure luck and that their existence has only limited impact on animal kingdom. We are, however, convinced that without the slow development, and in its reality extremely complex array of immune reaction, the Earth would still be covered by primordial soup full of prokaryotic animals. We have to remember that regardless of any horizontal exchange of biological information which might play a role in the evolution of immunity, there is no function without structure, and there is no structure without a history.[7] The immune strategy represents an appropriate morpho-functional pattern determined by a common body plan which itself has emerged during the evolutionary process, mostly under the pressure of the natural forces of environment.