Analysis of elevated serum interleukin-6 levels in rheumatoid arthritis: correlation with erythrocyte sedimentation rate or C-reactive protein.

The purpose of this study was to determine whether selected antirheumatic drugs would suppress elevated circulating interleukin-6 (IL-6) levels in patients with rheumatoid arthritis (RA). The 267 patients who enrolled in a double-blind randomized protocol received placebo, naproxen (1500 mg/day), or prinomide (1500 mg/day) for up to 16 weeks. Serum samples from 143 of the patients completing the trial and from 135 normal donors were analyzed by quantitative sandwich enzyme-linked immunosorbent assay for IL-6 concentrations. A mean normal IL-6 value was determined to be 3 pg/ml (95th percentile value = 10 pg/ml). IL-6 levels at baseline for the patients with RA were significantly higher than those for control subjects (p < 0.0001). Elevated IL-6 levels (> 10 pg/ml) at baseline were found in 80% of subjects with RA (median = 36 pg/ml, range 12 to 403). For patients with elevated levels of either IL-6, C-reactive protein (CRP), or erythrocyte sedimentation rate (ESR) at baseline, initial median values of IL-6, CRP, and ESR were compared with those from the final visit for each treatment group. There was no significant decrease in IL-6 levels with treatment. Median CRP levels decreased significantly, from 1.9 to 0.8 mg/dl (p = 0.002), as did median ESR (37 to 34 mm/hr, p = 0.013), only in the prinomide-treated group.

Inhibitory effect of sera from rheumatoid arthritis patients and enhancing effect of levamisole on total E-rosette levels of lymphoid cells from normal and RA subjects.

We determined the E-rosette levels (E-R) in rheumatoid arthritis patients (RA) and repeatedly tested a selected group with depressed E-R. We also evaluated, in vitro, the inhibitory effect of RA sera on normal E-R and the enhancing effect of levamisole (LV) on normal and RA E-R. Selection criteria and E-R protocol were those of Di Perri (1979). Mononuclear cells (MNC) from 23 normal donors had a mean E-R of 53 +/- 7% (X +/- SD) and this was unchanged after incubation with 32 microM LV. MNC from 31 RA displayed a value of 42 +/- 7% which was lower (p = 0.01) than the normal values and which increased to 47 +/- 9% after LV treatment (p = 0.02). Twenty of these RA did not show enhanced E-R after LV; the remaining 11 RA had E-R of 37 +/- 6% which were restored to 50 +/- 8% after incubation with LV (p = 0.001). We defined this latter group of RA as depressed responders (DR). Four DR were tested repeatedly over a four-month period and consistently remained depressed. Their sera had inhibitory activity on E-R of healthy donors. In vitro treatment of healthy MNC with these RA sera plus 3.2 microns LV abrogated the inhibitory effect. Both the IgG and the IgM fractions of the RA sera manifested this inhibitory effect. We conclude that RA can have depressed E-R which are not transient and can be corrected by in vitro incubation with an immunomodulator. Sera from these RA can inhibit E-R of normal MNC, and LV can abrogate the inhibitory activity of such sera.

The action of human and rabbit serum phospholipase A2 on Escherichia coli phospholipids.

We have compared the properties of phospholipase A (E.C. 3.1.1.4) activity in whole human and rabbit serum toward the phospholipids of Escherichia coli. Using as substrate E. coli labeled during growth with either [1-(14)C]-palmitic acid or [1-(14)C]oleic acid, and then autoclaved to inactivate E. coli phospholipases and to render the labeled phospholipids accessible to exogenous phospholipases, we show that the deacylating activity in both human and rabbit serum is almost exclusively of the A(2) type. Rabbit serum is at least 20-fold more active than human serum. Activity in both sera is maximal at physiological Ca(2+) concentrations (2 mM) and is abolished by ethylenediaminetetraacetic acid. To examine hydrolysis of intact (unautoclaved) E. coli treated with 25% serum, use was made of a phospholipase A-deficient E. coli strain (E. coli S17), thereby eliminating the possible contribution of bacterial phospholipases to degradation. Human and rabbit serum are about equally bactericidal toward E. coli and cause comparable structural damage. However, only rabbit serum produces substantial hydrolysis of the phospholipids of intact E. coli S17. Heated (56 degrees C, 30 min) rabbit serum is non-bactericidal and retains phospholipase A(2) activity toward autoclaved, but not intact E. coli. The ability of heated serum to degrade phospholipids of intact E. coli S17 is restored, however, by adding 25% normal human serum, which is bactericidal. In this combination, doses of heated rabbit serum containing as much phospholipase A(2) activity (toward autoclaved E. coli) as is present in 25% unheated rabbit serum, produce roughly the same extent of hydrolysis of intact E. coli as does normal rabbit serum alone. Low doses with a phospholipase A(2) activity comparable to that of normal human serum elicit little or no hydrolysis. These findings indicate that hydrolysis of the phospholipids of intact E. coli S17 by serum occurs when: 1) the serum is bactericidal, and 2) when sufficient phospholipase A(2) is present. The difference in phospholipid hydrolysis that accompanies killing of E. coli by human or rabbit serum appears to reflect, therefore, the different amounts of phospholipase A(2) activity in the two sera. Phospholipid degradation is not required for the bactericidal action of serum. Bacterial phospholipid breakdown may be important, however, in the overall destruction and digestion of invading bacteria by the host.-Kaplan-Harris, L., J. Weiss, C. Mooney, S. Beckerdite-Quagliata, and P. Elsbach. The action of human and rabbit serum phospholipase A(2) on Escherichia coli phospholipids.

Resistance of gram-negative bacteria to purified bactericidal leukocyte proteins: relation to binding and bacterial lipopolysaccharide structure.

The sensitivity or resistance of gram-negative bacteria to antibacterial systems appears to be related to the length of the saccharide chain of the bacterial envelope lipopolysaccharides (LPS). To explore this relationship further, we made use of two bactericidal, membrane-active cationic proteins, recently purified to near homogeneity, one from human and one from rabbit polymorphonuclear leukocytes (PMN). We have studied the effects of these two closely similar proteins on strains of Salmonella typhimurium and Escherichia coli, each separate strain differing in the saccharide chain length of its outer membrane LPS. Binding of these proteins to the bacterial outer membrane is required for killing, and is accompanied by an almost immediate increase in outer membrane permeability to normally impermeant actinomycin D. Sensitivity to the bactericidal and permeability-increasing activities of the human and rabbit proteins increases with decreasing LPS-saccharide chain length (chemotype: [S < Ra < Rb(3) < Rc < Rd(1)]). S. typhimurium G-30 and E. coli J5, mutant strains lacking UDP-galactose-4-epimerase, synthesize incomplete LPS (chemotype Rc) when grown without galactose, and are then as sensitive to both PMN proteins as the S. typhimurium strains 395 R10 (Rd(1)) and R5 (Rb(3)). However, when these mutants are grown with galactose, they synthesize complete LPS (chemotype S) and exhibit nearly the same relative insensitivity as the smooth strains S. typhimurium 395 MS and E. coli 0111:B4. The differences among strains in sensitivity to the effects of the proteins on bacterial viability and permeability correspond to differences in bacterial binding of these PMN proteins. Thus, at protein concentrations that produce maximal antibacterial activity toward the rough bacteria, but little or no activity toward the smooth strains, rough bacteria bind from 3- to 10-fold more protein (S. typhimurium 395 R10; S. typhimurium G-30, and E. coli J5 [grown without galactose]) than do the smooth bacteria (S. typhimurium 395 MS; E. coli 0111:B4; S. typhimurium G-30 and E. coli J5 [grown with galactose]). These findings suggest that bacterial sensitivity or resistance to these purified bactericidal PMN proteins is determined by the binding properties of the outer membrane, which in turn depends upon the LPS-saccharide chain length.

Separation and purification of a potent bactericidal/permeability-increasing protein and a closely associated phospholipase A2 from rabbit polymorphonuclear leukocytes. Observations on their relationship.

Two antibacterial proteins from rabbit polymorphonuclear leukocytes, a potent bactericidal cationic protein that increases the envelope permeability of susceptible gram-negative bacteria and a phospholipase A2, have been purified to near homogeneity by ion exchange, gel filtration, and hydrophobic interaction chromatography. The apparently noncatalytic bactericidal/permeability-increasing protein has an approximate molecular weight of 50,000 and is isoelectric at pH 9.5 to 10.0. The molecular properties, including amino acid composition, and the antibacterial potency and specificity of this rabbit leukocyte protein and of the bactericidal/permeability-increasing protein from human granulocytes that we have recently purified (J. Biol. Chem. 253, 2664-2672, 1978) are closely similar. Both proteins kill several strains of Escherichia coli and Salmonella typhimurium. Rough strains are more sensitive than smooth strains. All gram-positive bacterial species tested are insensitive to high concentrations of either rabbit or human protein. The phospholipase A2, purified by hydrophobic interaction chromatography on phenyl-Sepharose, ran as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 14,000 and had a specific enzymatic activity comparable to that of purified phospholipases A2 from other sources. Separation of the phospholipase A2 from the bactericidal/permeability-increasing protein has no noticeable effect on the bactericidal and permeability-increasing activities of the purified bactericidal protein, but removes the ability of the phospholipase A2 to hydrolyze the phospholipids of intact Escherichia coli. Upon recombination of the phospholipase A2 with the bactericidal/permeability-increasing protein, the phospholipase A2 regains its activity toward the phospholipids of intact E. coli suggesting that these two antibacterial leukocyte proteins act in concert.

Effects of human and rabbit serum on viability, permeability, and envelope lipids of Serratia marcescens.

The major action of serum on gram-negative organisms is thought to be on the microbial envelope. We compared the effects of normal human and rabbit serum on the envelope lipids of two strains of Serratia marcescens, one sensitive and one resistant to the bactericidal effects of serum. During killing by either serum, the sensitive strain underwent rapid permeability changes coincident with degradation of microbial phospholipids. The resistant strain exhibited none of these effects. The phospholipid degradation that accompanies killing of the sensitive strain by serum could be caused by phospholipases present in serum or by Serratia's own phospholipid-splitting enzymes. The results indicate that phospholipid breakdown is caused by activation of bacterial of bacterial phospholipases and not by serum phospholipases. This conclusion is based upon the following findings.(i1 Although rabbit serum phospholipase A was at least 10 times more active than human serum phospholipase A, phospholipid degradation in the sensitive Serratia strain was comparable during (equally rapid) killing by human or rabbit serum. (ii) Heat treatment (56 C) of both sera eliminated bactericidal activity as well as microbial lipid degradation but abolished phospholipase activity of human serum only. (iii) Virtually complete removal of phospholipase A activity from human serum by adsorption onto autoclaved Micrococcus lysodeikticus had no effect on the extent of phospholipid hydrolysis or on bactericidal activity. Activation by serum of endogenous phospholipase activity in S. marcescens was accompanied by enhanced incorporation of lipid precursors into bacterial lipids. No evidence was found for increased turnover of protein or ribonucleic acid during killing by serum.