Modulation of functions of granulocytes by recombinant human GM-CSF and possible complications of GM-CSF therapy.

Besides its effect on bone marrow progenitors, GM-CSF is able to modulate functions of mature cells such as neutrophils. It inhibits random migration and chemotaxis through action on both cells and chemotactic factors, and stimulates oxidative metabolism as well as elastase release. Furthermore, it strongly enhances the response of the cells to the usual stimulants such as f-Met-Leu-Phe and phorbol esters. The role of neutral proteinases and activated oxygen species in different diseases such as ARDS, emphysema, coagulation defects, arthritis, and inflammation, is recognized. The remarkable in vitro release of neutral proteinases and activated oxygen species from granulocytes after GM-CSF stimulation may be of importance in vivo. This should be considered in clinical application of GM-CSF, particularly with high-dose therapy.

A new method for fluorescence immunoassay using plane surface solid phases (FIAPS).

A method distinguished by high sensitivity, low non-specific binding, easy handling, and broad applicability with respect to various antigens is described. Films of polymethyl-methacrylate with plane surfaces were selected as solid phase for adhesive or covalent binding of different antigens (DNA, histone, human, rabbit or goat immunoglobulins). Proteins were covalently bound to the films by the azide method (Orth and Brummer, 1972). Polymethylmethacrylate films thus coated had a negligible autofluorescence and gave minimal non-specific binding of protein. Coated films were used for specificity control of FITC-labeled antibody preparations and in the double antibody and sandwich techniques for detection of antibodies or antigens in sera from man, rabbit and goat. FITC-conjugated hyperimmune antibody, in some cases purified by immunoadsorption was used as second antibody in indirect techniques. The amount of fluorescent-labeled antibody bound per unit of surface area of film was measured by incident light with a Zeiss-Axiomat fluorescence microscope equipped for fluorescence photometry and an uranyl acetate glass plate was used as a standard. The technique appears superior to present methods of quantitative immunofluorescence analysis.

Effect of granulocyte-macrophage colony-stimulating factor on the induction of unresponsiveness by lymphoid cells.

Skin grafts can be significantly prolonged in ALS-treated mice by the injection of 25 x 10(6) donor bone marrow cells or 50 x 10(6) spleen cells. Lymph node cells and thymocytes are only minimally effective in prolonging grafts. The effect of a hematopoietic growth factor, granulocyte-macrophage colony stimulating factor (GM-CSF) was studied in this model of unresponsiveness. C3H/He lymphoid cell donors were treated with GM-CSF. Either normal or GM-CSF-treated cells were injected into ALS-treated B6AF1 mice grafted with C3H/He skin. GM-CSF treatment significantly augmented the effect of marrow in prolonging graft survival at doses of 1 to 25 x 10(6) cells. In contrast, GM-CSF had no effect on the graft-prolonging effect of spleen cells when 50 x 10(6) cells were given. When the dose of cells was reduced to 25 x 10(6), graft survival in the group given GM-CSF-treated cells was prolonged compared with survival in the group given normal cells. Grafts in the group given GM-CSF-treated lymph node cells were rejected in sensitized fashion. When marrow and spleen are separated on a Percoll gradient, the cell active in promoting graft survival is recovered primarily in the 52.5% fraction. The graft-prolonging effect of the 52.5% marrow fraction was not affected by GM-CSF treatment. In contrast, GM-CSF-treated marrow cells in the 60% fraction significantly prolonged graft survival, while normal marrow cells in this fraction had no effect on graft survival. GM-CSF-treated spleen cells in the 52.5% and 60% fractions significantly decreased graft survival compared with normal cells when given at a dose equal to the number of cells recovered from 50 x 10(6) cells. When the dose of fractionated spleen cells was reduced, GM-CSF-treated spleen cells were more effective than normal cells in prolonging graft survival. These results indicate that GM-CSF activates a cell in marrow that promotes graft survival. This cell is recovered in the 60% Percoll fraction. In contrast, GM-CSF appears to affect two cell populations in spleen, one beneficial and one detrimental to graft survival. The predominant effect depends on the dose of spleen cells that is given.

In-vivo phagocytosis: enhancement of bacterial clearance by native and enzyme-treated immunoglobulins.

The enhancement of bacterial blood clearance in mice by native and enzymatically derived fractions of rabbit anti-E. coli hyperimmune serum was tested. Native immune serum, the corresponding IgG, F(ab')2 and Facb fractions strongly augmented the phagocytosis rate of bacteria, whereas Fab/Fc, Fab, Fc fragments, corresponding preparations from normal serum, and E. coli-absorbed preparations showed no marked enhancing capacity. In one experiment opsonization by IgM was demonstrable. Mice that were injected with fatal doses of bacteria could be protected by subsequent treatment with IgG or F(ab')2 preparations of rabbit anti-E. coli serum. Conclusion is drawn that the Fc region of the IgG molecule is not predominantly responsible for opsonized clearance while the intact divalent antigen cross-linking F(ab')2 fragment - presumably by virtue of complement activation via the alternate pathway - could mediate enhanced bacterial clearance.

Mouse spleen cells are sensitized sheep red blood cells: an Fc-rosette-forming system allowing the detection of "activated" Fc structures.

From a variety of Fc receptor-bearing cell/sensitized red blood cell combinations, mouse spleen cells, and sensitized SRBC were selected as an Fc-specific EA rosette assay system because only this mixture combined a high percentage (about 50%) of rosette-forming cells with complete absence of spontaneous rosettes and showed no influence of complement on the rosette formation. From studies on the minimal structural requirement of IgG both for mediation and inhibition of EA rosettes using IgG and several well-defined fragments, it appeared that both the CH2 and the CH3 domain of Fc are needed for optimal interaction with the lymphocyte Fc receptor. Finally, it was demonstrated that the assay system is able to detect "activated" Fc structures (here: heat-aggregated IgG) and to differentiate between varying amounts of such structures.

The modulation of immune complex aggregation by classical pathway-mediated reactions.

Classical pathway (CP)-triggered reactions of complement-modulated immune complex (IC) aggregation (tetanus toxoid/human anti-tetanus toxoid-IgG; ICs of equivalence) were investigated turbidimetrically during the early stages of reaction. Monospecific Fab'- or Fab-fragments (rabbit) directed against certain complement components were used to block the complement function in normal human serum (NHS). Additionally, parts of the reactions were studied using purified complement components. C1q in serum generated by the addition of EDTA as well as purified C1q were found to increase the IC aggregation. In contrast to C1q, macromolecular C1 is able to inhibit IC aggregation, whereas additional participation of C-1 INH reversed this process. The cooperation of the remaining CP proteins (C4, C2, C4bp, and I) reconstituted the inhibition capacity of the complement. Whereas C3 supported significantly inhibition, a significant influence of other effector pathway (EP) components (C5-C9) was not detectable turbidimetrically.

The functional inhibition of activated C1 inhibitor in normal human serum causes spontaneous consumption of the complement components C2, C3, C4, and factor B.

The human complement components C1r, C1s, C4, C3, factor B, and/or activated C1INH were functionally blocked in normal human serum (NHS) and EGTA- or EDTA-treated NHS by polyclonal monospecific Fab'-fragments to the individual components. The results of inhibition experiments are compatible with the formation of a classical pathway fluid-phase C3 convertase (C4b2a) spontaneously generated by the inhibition of activated C1INH. This process in both NHS and EGTA-NHS was accompanied by the consumption of C2, C4, C3, and factor B but only by poor enhancement of C5 conversion. Blocking subcomponent C1r, completely inhibited spontaneous activation of the complement components, indicating that the control of C1r hydrolysis is the essential role of activated C1INH as a regulator of C1 activation in NHS. Non-complement serum proteases were inactive during the initiation of the activation process. The presence of blood cells during functional inhibition of activated C1INH in NHS slightly decreased the consumption of C3 but not of C2 and C4.

BCG versus VCN: the antigenicity and the adjuvant effect of both compounds.

Enzymatically active Vibrio cholerae neuraminidase (VCN) acts as an adjuvant when it is combined with an antigen. This has been demonstrated for bacterial, viral, protein, and cellular antigens and was found for the humoral, but more pronounced for the cellular immune response. In this study, the adjuvant activity of VCN for live BCG is discussed. Live BCG itself is regarded as a rather potent immunostimulator. A total of 6.5 mU of VCN, being most effective as derived from previous adjuvant experiments, was combined with 10(6) or 10(7) live BCG and injected IV into mice. Booster injections were given on day 12. Challenge was administered in the footpad on day 17 either with 6.5 mU VCN or with 5 x 10(4) or 5 x 10(6) dead BCG. Footpad swelling was recorded 24 h after challenge. After clinical examination, mice were bled and antibodies against BCG and VCN were measured. The results show that VCN specifically enhances the cellular, not the humoral, immune reaction against BCG, while BCG is unable specifically to enhance immune reaction against VCN. However, BCG unspecifically stimulates the cellular reaction of the organism against an antigen, primarily injected, subsequent to BCG for instance, against VCN.

Haematological effects of rhGM-CSF in dogs exposed to total-body irradiation with a dose of 2.4 Gy.

It was the specific aim of this study to test the stimulatory effects of recombinant human GM-CSF (rhGM-CSF) on haemopoietic regeneration in dogs which had received total-body irradiation (TBI) with a dose of 2.4 Gy. In normal dogs rhGM-CSF given subcutaneously at 10 microgram/kg per day or 30 microgram/kg per day for 21 days caused strong but transient increases in the peripheral blood neutrophils. The monocyte counts also showed a transient rise during treatment in a dose-dependent fashion, whereas the lymphocyte counts increased only at the higher dose of rhGM-CSF and the platelet counts were transiently depressed during the course of the treatment. In the irradiated animals treatment with rhGM-CSF decreased the severity and shortened the duration of neutropenia but had no significant influence on monocyte or lymphocyte recovery. The granulocyte values showed a characteristic pattern of fluctuations with the first peak occurring at the same time (day 10 to day 13) when the abortive rise was observed in the untreated dogs. In contrast the GM-CFC in the peripheral blood remained depressed during the whole treatment course, similar to the untreated irradiated controls. These results indicate that treatment with GM-CSF can be an effective biological monotherapy for radiation-induced bone marrow failure, but that for higher radiation doses the number of GM-CSF responsive target cells will become a critical determinant of therapeutic efficacy.

Combined effects of granulocyte-macrophage colony stimulating factor with erythropoietin in subhuman primates.

Recently, we showed in a cynomolgus monkey model that the combination of interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factors (GM-CSF) demonstrates synergistic effects, if administered sequentially. Not only were progenitor cells of the myelocytic lineage stimulated cooperatively, but platelet release was also observed. These effects could not be found using these factors alone. In this study, GM-CSF was administered with erythropoietin (EPO) in a sequential manner. This treatment of GM-CSF and EPO resulted in a GM-CSF-stimulated increase in cells of myelomonocytic differentiation, and enhanced stimulation of erythroid and megakaryocytic lineages. The observed effects of growth factor combination were seen to the same extent with intravenous or subcutaneous administration. The results obtained may predict the potential usefulness of GM-CSF treatment together with EPO. This could improve the effectiveness of single growth factors in only limited indications, e.g., reduced need for transfusion (platelets, blood), correction of iatrogenic anemia, and recovery from myelosuppression due to chemo- and radiotherapeutic agents.

Therapeutic effects of 15-deoxyspergualin in acute and chronic relapsing experimental allergic encephalomyelitis (EAE) as models for multiple sclerosis (MS).

Experimental allergic encephalomyelitis (EAE) is an inflammatory and paralytic auto-immune disease of the central nervous system (CNS) resulting from the sensitization of susceptible laboratory animals with whole brain homogenate, lyophilized spinal cord or heterologous myelin basic protein (MBP) in complete Freunds adjuvant (cFA). Acute EAE generally presents as a monophasic disease that produces perivascular inflammation and sometimes small areas of demyelination. The chronic relapsing form of EAE in particular offers important similarities to the human disease multiple sclerosis (MS). Due to its immunosuppressive mode of action, the authors wished to examine the therapeutic effects of 15-deoxyspergualin (15-DOS) in the two models of acute and chronic relapsing EAE in Lewis rats. In the first model, sensitization of adult rats (8-12 weeks of age) with guinea pig spinal cord in cFA results in an acute clinical episode of severe EAE, and by day 17 all animals died. Lewis rats treated with 15-DOS showed a delayed and reduced onset of clinical symptoms and even low amounts of the drug prevented mortality. The protection afforded by 15-DOS was long-lasting and no subsequent relapse has been observed. On rechallenging, such convalescent rats were totally resistant to re-induction of the disease. Passive transfer of EAE to naive Lewis rats was possible with encephalitogenic (antigen-specific primed) spleen cells from diseased control animals but could be prevented by fresh 15-DOS treatment. Passive transfer of acute EAE was suppressed when spleen cells from diseased Lewis rats but 15-DOS-treated animals were used.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunosuppressive activity of 15-deoxyspergualin (15-DOS) on various models of rheumatoid arthritis.

Based on the promising results obtained thus far in various auto-immune disease models, the authors have further elucidated the disease-modifying potential of the new immunosuppressive drug 15-deoxyspergualin (15-DOS) in models of inflammatory and chronic degenerative joint disease of adjuvant arthritis (AA) and collagen type-II induced arthritis (CIA) in Lewis rats and spontaneously developing polyarthritis in MRL/1 mice. Treatment of AA animals with 15-DOS starting with the day of adjuvant injection prevented the disorder from spreading to the non-injected extremity. The drug also reduced the arthritis index (47% inhibition). The adjuvant disease can thus be prevented nearly totally, even after discontinuation of drug application. Even when starting the therapy during the established disease, 15-DOS still exerts a protective action on the development of the adjuvant disease. In the present model of CIA, the animals showed the same progression as reported by others. Approximately 80% of the animals had bilateral hind paw swelling. The production of autoreactive anti-type-II collagen antibodies and also high levels of circulating rheumatoid factor (RF) can be demonstrated in the serum of CIA rats. Treatment of these animals with 15-DOS was very effective in inhibiting swelling of the joints. Average antibody titres were also depressed and circulating RF was reduced. Even when treatment with 15-DOS started on day 15 after CIA induction on established lesions of polyarthritis, not only a significant suppression of the paw diameters occurred, but also the humoral response (antibody formation) could be inhibited in animals with established CIA disease. The disorder of MRL/1 mice is characterized by the development of auto-antibodies most prevalently directed against double-stranded DNA (dsDNA) and type-II collagen. These mice also have circulating rheumatoid factor (RF) and develop histological changes in their joints characterized by pannus formation, cartilage and bone erosions. Treating MRL/1 mice with 15-DOS resulted in a decrease in the amount of auto-antibodies and inhibited developing polyarthritis. Even in an established disease, 15-DOS reduced circulating RF and increase in paw volume (signs of polyarthritis) was inhibited. These results suggest that 15-DOS might be used as a therapeutic agent for human RA and that this new immunosuppressive drug could be an effective nonsteroidal antirheumatic agent.

A rabies-specific human monoclonal antibody that protects mice against lethal rabies.

According to a recommendation from WHO (World Health Organisation) for prevention of a possible rabies infection, active vaccination has to be combined with application of immunoglobulin to get a fast protective effect. At present, preparations of purified human or equine rabies-specific immunoglobulin are used. We have generated a human rabies-specific monoclonal antibody (huMAb) by immortalization of human B-cells with Epstein Barr Virus (EBV), followed by fusion with a mouse myeloma cell. The resulting clone TW-1 secrets an IgG1 lambda huMAb which specifically reacts in ELISA with 5 laboratory rabies virus strains of serotype 1 and DUV3 (Duvenhage, serotype 4). Western Blot analysis revealed fine specificity for the G glycoprotein (gp67) of rabies virus. HuMAb TW-1 neutralizes rabies virus in vitro (RFFIT) as well as in vivo and protects rabies infected mice. Compared to polyclonal human rabies immunoglobulins, huMAb TW-1 is advantageous, because of its defined specificity and the very low amounts of total protein needed for therapeutic effects.