Expression of an immunoglobulin heavy chain transgene in macrophage as well as lymphocyte lineages in vivo.

A rearranged immunoglobulin heavy chain (IgH) transgene-encoded protein is expressed in macrophage lineage cells, in addition to B and T lineages, in transgenic mouse bone marrow. Peripheral macrophages also express transgenic IgH protein. Mature T cells express lower levels than immature thymocytes. Almost all B220+ cells in the bone marrow express transgenic IgH protein, and this early expression in the B lineage is accompanied by a reduction of cell frequency even in the early B220+ CD43+ BP-1- stages, although it is more prominent in BP-1+ pre-B cells. Thus, an IgH transgene can be expressed not only in lymphoid but also in myeloid cells, although its developmental effects are restricted to the B cell lineage.

Presence of activated antigen-binding B cells during immunization enhances relative levels of IFN-gamma in T cell responses.

To examine the influence of Ag presentation by B cells on immune responses, we have used mice transgenic for an Ig heavy chain from a monoclonal anti-azobenzenearsonate (Ars) Ab to deliver Ag to B cells during immunization. A large proportion of transgene-expressing B cells in these mice binds Ars, while transgenic serum Ig shows poor Ars binding. Transgenic B cells present Ars proteins better than their nonhaptenated counterparts. This is associated with an increase in the proliferative responses of transgenic T cells to Ars protein immunization. Although B cell numbers in the transgenic mice are lower, many B cells in them show an activated phenotype, as identified by altered surface levels of peanut agglutinin reactivity, CD23, CD24, CD44, CD62L, and CD86. Even against nonhaptenated immunogens, transgenic responses show significant enhancement in the relative proportions of the Th1 cytokine IFN-gamma over the Th2 cytokines IL-4 and IL-10. Haptenated immunogens further enhance the predilection of transgenic mice to produce relatively more IFN-gamma. Consistent with this, there is an increase in IgG2a/IgG1 ratios in serum Abs in response to haptenated immunogens in transgenic mice. Adoptive transfer of primed hapten-specific secondary B cells into nontransgenic mice also induces an increase in relative levels of IFN-gamma in response to haptenated immunogens. Thus, presentation of immunogen in vivo by activated Ag-binding B cells contributes to enhanced immunogenicity and a Th1 cytokine bias.

Virus-induced leukopenia: Colorado tick fever as a human model.

Eight patients with Colorado tick fever were studied to determine whether alterations in the production of granulopoietic stimulatory or inhibitory factors (or both) could be found in association with the leukopenic state of the disease. The studies demonstrate that in the patients with Colorado tick fever the mononuclear cell production of colony-stimulating factor is decreased and that there is an increase in circulating inhibitory factors in the serum of such patients. The depressed mononuclear cell colony-stimulating activity does not appear to be reversible by addition of either endotoxin or normal human serum. Characterization of these serum inhibitory factors may facilitate understanding of leukopenia in human disease.

Longterm effects of cyclophosphamide on granulocyte colony formation in patients with rheumatoid arthritis.

Longterm cytotoxic therapy, particularly with alkylating agents, is frequently associated with the development of second neoplasms, particularly acute myeloid leukemia. Twelve patients with classic, progressive rheumatoid arthritis (RA), treated with a cytotoxic agent, cyclophosphamide, from 1969 to 1974 at the University of Colorado Health Sciences Center were reassessed for current status of the granulocytic system. Bone marrow biopsies were investigated histologically and the number of granulocyte precursor cells judged using colony forming cells in culture (CFU-C). Control bone marrow biopsies were done on 8 patients with classic, progressive RA who had not taken cyclophosphamide as well as 16 healthy controls. Colony forming cell numbers in patients with RA who had been treated with cyclophosphamide were significantly lower compared to patients with RA who had not received cyclophosphamide and normal controls. These data suggest that cyclophosphamide may cause suppression of the hematopoietic system that does not totally recover even after long periods of time.

Schistosomal stimulation of eosinophil production by human bone marrow in vitro.

These studies were undertaken to determine if schistosomal and ragweed antigens incubated with human peripheral blood mononuclear cells (MNC) would stimulate the production of an eosinophil colony stimulating factor (CSF) which was active on human bone marrow. These studies have shown that schistosoma mansoni antigen in a concentration of 5 micrograms/ml incubated with MNC's from unsensitized individuals lead to production of a conditioned medium which did not stimulate an increased number of total colonies but did increase the number of eosinophil colonies (44 to 61 colonies). Ragweed antigen also stimulated increased eosinophil CSF (from 44 to 63 colonies). The granulocyte response to parasitic antigens almost invariably involves the eosinophil system. Eosinophils play a vital role in the control of these diseases in humans. Further understanding of the interaction of parasitic antigens with the granulopoietic system will hopefully give new and important clues as to how such parasites interact with man and how they are controlled.

Urinary granulocyte colony-stimulating factor in bilharziasis.

Studies have been done to determine the levels of human urinary granulocyte colony-stimulating factor in Egyptian patients with active bilharziasis. Colony-stimulating factor levels were measured by a semi-solid tissue culture colony assay with murine bone marrow as the target cell source. The levels in urine from patients with bilharziasis (mean 118) were found to be significantly elevated above control values found in normal human urine (mean 72) derived from the same population. This is the first demonstration of an effect of parasitic infection in man on the granulocyte regulatory system, and opens the way for future studies in this area.

Regulation of granulopoiesis and distribution of granulocytes in early phase of bacterial infection.

Studies have been carried out to determine the effect of bacterial infection on CSF production, CFU-C activation, and bacterial clearance by mature granulocytes in mice infected with Escherichia coli. These studies have shown that immediately after bacterial infection (5 minutes), serum colony-stimulating factor (CSF) levels and bone marrow colony-forming units in culture (CFU-C) levels are elevated. This is followed by oscillatory rises in both of these parameters and the appearance of granulocytes in the infected site. With clearance of bacteria, CSF and CFU-C levels return to normal. These studies have indicated further that bacterial infection is a major stimulus for granulocyte production through the CSF-CFU-C system and that clearance of bacteria by mature granulocytes may serve as a negative feedback regulatory arm.

[Bacterial infection and the regulation of in vivo granulopoiesis]. / Bakterielle Infektion und Regulation der Granulopoiese in vivo.

The mechanism which regulates granulopoiesis was investigated in C 57 BL mice injected i.p. with E.coli. The positive and negative feedback arm of the regulation was studied by correlating the number of bacteria, the number of granulocytes, serum CSF levels and the number of CFU-C in the bone marrow.

Colony stimulating activity in normal human serum tested against human bone marrow.

Two hundred and thirty normal human sera have been tested for colony stimulating factor (CSF) under carefully controlled conditions using human bone marrow and the semi-solid agar-gel technique. It has been shown that CSF can be detected in most sera when incorporated into underlayers to remove potential inhibitors. The effect of storage temperature on serum CSF has been determined. Storage at 4 degrees, --15 degrees, and --66 degrees for up to 80 days resulted in only a mild decrease in CSF levels. After 240 days of storage, CSF values fell to 50% of that found before storage. Repeated freeze-thawing of serum has not been shown to decrease CSF levels when done in Pyrex glassware. These studies will serve as a baseline for those wishing to study human serum CSA in hematopoietic disorders.

In vivo and in vitro effects of lithium on granulopoiesis in human neutropenic disorders.

Studies have been cazrried out to determine the in vivo and in vitro effects of lithium carbonate in various neutropenic disorders. Addition of lithium to culture of bone marrow from patients with a variety of neutropenic disorders and ANLL in which normal or elevated serum and/or urinary CSA levels were present did not enhance granulocyte colony formation. Administration of lithium carbonate to patients with Felty's syndrome, in which serum and urinary CSA levels are reduced, enhanced peripheral blood neutrophil levels and serum and urinary CSA values. Lithium administration appears to be most beneficial in neutropenic patients in whom it can be demonstrated that CSA production is reduced.

Recovery of CFU-C after freezing of normal and leukemic human bone marrow.

Studies have been carried out to determine the viability of leukemic and normal human bone marrow, cryopreserved in liquid nitrogen at -196 degrees C, using changes in total cell numbers and granulocyte colony forming ability in vitro. These studies have shown that there is considerable variability in the recovery of CFU-C from individual specimens. When the overall recovery, in all patients, is taken into account, there is a gradual decline in CFU-C numbers to about 60% after 24 months of freezing. CFU-C recovery is closely correlated with recovery of total cell numbers.

Autologous bone-marrow and peripheral blood buffy coat cell infusion in the treatment of chronic myeloid and acute leukemia.

Autologous marrow infusion has been attempted in three patients with chronic myeloid leukemia (two in blast crisis, one with severe myelofibrosis and pancytopenia) and one patient with acute lymphatic leukemia. One patient with blast crisis of CML expired prior to marrow infusion. One patient with myelofibrotic phase of CML is alive seven months post marrow infusion. The other two patients expired 6 and 16 days post marrow infusion. Bone-marrow repopulation is feasible in the face of severe myelofibrosis.

Bacterial stimulation and granulocyte inhibition of granulopoietic factor production.

We attempted to determine the effect of live bacteria (Staphylococcus epidermidis) on granulocyte colony-stimulating-factor production by human peripheral blood mononuclear cells (monocytes and lymphocytes) in vitro. Addition of bacteria to mononuclear-cell cultures enhanced colony-stimulating-factor production by these cells, as assayed on both human and mouse bone marrow. Addition of peripheral blood granulocytes to parallel cultures eliminated this enhancement effect, presumably by bacterial removal or inactivation. These data suggest that micro-organisms may have a pivotal role in granulocyte production and maturation by serving as a stimulus to increase colony-stimulating-factor production and also as negative control through their removal by the newly formed granulocytes.

Colony growth of normal human bone marrow in agar-gel.

A study of the colony forming ability of 99 normal human bone marrows using the semi-solid agar technique is presented. Feeder layers of 10(6) normal human peripheral white blood cells were used as the stimulus, and were overlaid with 2 X 10(5) bone marrow cells. The presence of human plasma either in feeder layers or bone marrow overlays inhibited colony formation when compared with studies performed with cells washed free of plasma. The number of colonies grown varied, but not significantly when the same marrow was grown on feeder layers from different donors. The inhibitory effect of normal human plasma was also demonstrated when unwashed and washed bone marrow cells were plated over feeder layers with no stimulus. Further confirmation of the inhibition by normal human plasma was made in 10 cases by the addition of 0.1 ml plasma to cultures of washed marrow over washed feeder layers. These data suggest that optimum results from in vitro culture of normal human bone marrow can be obtained only when all plasma is washed from both the feeder layer cells and the marrow cells before culturing.